WO2008058897A2 - Diphyllin and other lignans as a medicament for v-atpase mediated disease - Google Patents

Diphyllin and other lignans as a medicament for v-atpase mediated disease Download PDF

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WO2008058897A2
WO2008058897A2 PCT/EP2007/062081 EP2007062081W WO2008058897A2 WO 2008058897 A2 WO2008058897 A2 WO 2008058897A2 EP 2007062081 W EP2007062081 W EP 2007062081W WO 2008058897 A2 WO2008058897 A2 WO 2008058897A2
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lignan
diphyllin
atpase
independently
acidification
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WO2008058897A3 (en
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Mette Grøndahl SØRENSEN
Kim Henriksen
Morten Asser Karsdal
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Nordic Bioscience A/S
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • Diphyllin and other lignans as a medicament for V-ATPase mediated disease
  • the present invention relates to a newly discovered activity of lignans including the compound diphyllin in proton pump blocking for use for instance in treating gastric disorders or for reducing the activity of osteoclasts.
  • Proton pump inhibitors such as omeprazole are a widely used class of pharmaceutical whose main action is pronounced and long-lasting reduction of gastric acid production. They have proved useful in the treatment of a range of conditions. They act by irreversibly blocking the hydrogen/potassium adenosine triphosphatase enzyme system (the H+/K+ ATPase, or more commonly just ⁇ gastric proton pump') of the gastric parietal cell. Cross-reactivity has previously been observed between V-ATPase (see below) blockers and gastric proton pump blockers 1 .
  • the proton pump is the terminal stage in gastric acid secretion, being directly responsible for secreting H + ions into the gastric lumen, making it an ideal target for inhibiting acid secretion .
  • Targeting the terminal-step in acid production, as well as the irreversible nature of the inhibition, results in a class of drugs that is significantly more effective than H2 antagonists and reduces gastric acid secretion by up to 99%.
  • V-ATPases Vacuolar H + -ATPases
  • the osteoclast V-ATPase contains an osteoclast specific subunit, the a3 subunit 11 ' 12 .
  • Loss of function mutations in the a3 subunit lead to severe osteopetrosis in both humans and mice, due to reduced acidification of the resorption lacuna 3 ' 5 ' 13 .
  • V-ATPase inhibitors are known, such as the natural compounds Bafilomycin Al 14 ' 15 and Concanamycin 15 as well as the synthetic inhibitors FR167356 16 and SB242784 17 . All these compounds potently inhibit osteoclastic resorption via inhibition of the acidification of the resorption lacuna, although the natural compounds are more than 100 fold more potent than their synthetic counterparts 14"17 .
  • Diphyllin (l-hydroxy-2- (hydroxymethyl) -6, 7-dimethoxy-4- (3, 4-methylenedioxy-phenyl) -3-naphthoic acid- ⁇ -lactone) was initially isolated as a natural compound found so far in five different plants Cleistanthus collinus, Justicia procumbens, Haplophyllum bucharicum, Justicia patentiflora and Phyllanthus polyphyllus 18'30 .
  • Diphyllin is a member of a family of lignan natural products also called phytochemicals, and diphyllin is a
  • Ramontoside is diphyllin-4-O-glucopyranosyl (1-4) -2, 3-di-0- methylxylopyranoside . Its structure is shown in Figure Ib. Cleisthanin A is shown in Figure Ic.
  • the lignan natural products and their associated glycosides are related with a range of biological activities, which include cytotoxic effects as well as antitumor activity and anti-inflammatory effects 18 ' 23"25 ' 27 ' 28 ' 33 ' 34 .
  • the present invention now provides a V-ATPase or gastric proton pump inhibiting lignan (or alternatively the use of a V-ATPase or gastric proton pump inhibiting lignan in the preparation of a medicament) for use in reducing the bone resorption activity of osteoclasts or in reducing gastric acidification, thus being useful in the treatment of excessive osteoclast action (e.g. loss of bone mass) or in the treatment of excess gastric acidification.
  • Said lignan is preferably of the general formula 1
  • R a is:
  • alkyl preferably Ci-6, alkaryl, in which the alkyl moiety is preferably Ci-6, aralkyl, in which the alkyl moiety is preferably Ci-6, e.g. benzyl, cycloalkyl, preferably C3-6, alkenyl, preferably C2-6 , carboxyalkyl in which the alkyl moiety is preferably Ci-6, alkoxycarbonyl, e.g. C1-4 alkoxycarbonyl, alkoxycarbonylalkyl, e.g. Ci- 4 alkoxycarbonyl-Ci- 4 alkyl, alkylcarbonyl, e.g.
  • Ci- 4alkylcarbonyl alkylcarbonylalkyl, e.g. C 1 - 4 alkylcarbonyl-Ci-4alkyl, carboxy, or OR a , wherein R a is as defined above, or alkylcarbonyloxy, e.g. C1-4 alkylcarbonyloxy;
  • R b and R c each independently is a group listed above in relation to R a , or together form an alkylene bridge, preferably a methylene bridge;
  • R d and R e each independently is a group listed above in relation to R a , or together form an alkylene bridge, preferably a methylene bridge;
  • R f independently is a group listed above in relation to R a , or OR f is replaced by H;
  • R g is H or is F, -CF 3 , -CH 3 , -00CH 3 , -OR h , or -SR h , where R h is H, or alkyl;
  • R 1 independently is a group listed above in relation to R a , for instance -OH, -OMe, or -Me; each dotted bond independently is present or absent; and wherein the lactone ring is optionally opened and is optionally esterified at the resulting hydroxyl and/or the resulting carboxylic acid functionality, but wherein R b and R c do not together form an alkylene bridge except when (a) R d and R e together form an alkylene bridge, or (b) wherein the dotted bonds are all present, or (c) wherein both (a) and (b) apply.
  • Said lignan may be of the general formula 2
  • the lignan may be of the general formula 3
  • R a ⁇ e are as defined above and each dotted bond is independently present or absent.
  • R d and R e form an alkylene, preferably methylene, bridge and in Formulae 1 and 2 that R f is H.
  • R f is H.
  • one or both of the dotted double bonds is present as a double bond.
  • the lignan may be of the general formula 4:
  • R a c are as defined above.
  • R b and R c are each preferably methyl, and R a is preferably hydroxyl or a glycoside group.
  • the active compound may be according to the general formula 5
  • a glycosylating moiety an amino glycosylating moiety alkyl (e.g. Ci-6 alkyl, especially methyl), alkaryl, e.g. Ci-6 alkylaryl, aralkyl, e.g. aryl Ci-6 alkyl (in each of which aryl may be phenyl), cycloalkyl, e.g. C3-6 cycloalkyl, alkenyl, e.g. C2-6 alkenyl, carboxyalkyl, e.g. carboxy Ci-6 alkyl, or alkylcarbonyloxy, e.g. C1-4 alkylcarbonyloxy, such as acetyl, or
  • R a is as defined above, e.g. is H (diphyllin) , or methyl (Justicidin A), or ethyl.
  • Suitable glycosylating moieties include ⁇ -D- apiofuranosyloxy, ⁇ -D-xylopyranosyl- (1 ⁇ 2) - ⁇ -D- apiofuranosyloxy, and ⁇ -L-arabinopyranosyl- (1 ⁇ 6) - ⁇ -D- glucopyranosyloxy, or the glcosylation seen in patavine 34 .
  • Suitable arylnaphthalene lignans include diphyllin, ethoxydiphyllin and other alkoxydiphyllins, acetyldiphyllin and other diphyllin esters, justicidin (A or B) , patavine, and cleisthanins (including A and B) .
  • podophyllotoxins have been found not to be active in the invention.
  • Examples of compounds for use in the invention include the following compounds tested in Example 8:
  • the invention includes these compounds per se.
  • the invention includes methods of treatment of V-ATPase or gastric proton pump mediated diseases comprising administering to a patient in need thereof a therapeutically effective amount of an active compound described herein.
  • Active compounds of the invention may be used in the treatment of excess gastric acidification or excess gastric acidification diseases including peptic ulcer disease, duodenal ulcers, reflux esophagitis, heartburn, Helicobacter pylori positive duodenal ulcer disease, Helicobacter pylori positive gastric ulcer disease, gastroduodenal ulceration associated with the use of non-steroidal anti-inflammatory drugs (NSAIDs) , acid-related indigestion (dyspepsia) , aspiration pneumonitis and rare illnesses such as Zollinger- Ellison syndrome, as well as inflammatory bowel diseases, such as Morbus Crohn (Crohn's disease) and Colitis Ulcerosa (ulcerative colitis) , and also in osteoclast mediated diseases, particularly those diseases in which osteoclast activity in re
  • Bone loss can also be a symptom associated with Crohn's disease and ulcerative colitis, or with the treatment of these and other conditions with corticosteroids, and can be treated according to the invention.
  • Treatment according to the invention may be for the relief of symptoms or may be preventative and administered in advance of symptoms appearing.
  • compounds of the invention may be administered orally or in some instances rectally, and may be formulated as tablets or capsules, optionally in enteric forms, including enterically coated granules within a capsule and multi- pellet unit forms, and/or slow release forms.
  • enteric forms including enterically coated granules within a capsule and multi- pellet unit forms, and/or slow release forms.
  • Alternative forms include chewable tablets and powders.
  • the compounds may also be formulated for administration parentally, including injectable forms for e.g. IV administration.
  • the compounds of the invention may be incorporated in a nutraceutical product which may take the form of a solid foodstuff such as a cereal bar or a liquid or infusible product for making a drink, possibly a hot drink such as a ⁇ tea' .
  • the invention therefore includes a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising an active compound of the invention mixed with a pharmaceutically or veterinarily acceptable carrier or excipient material, said composition preferably being a solid composition.
  • Suitable dosage levels of the active will be from 0.01 to 1000 mg/kg, more preferably 0.1 to 100 mg/kg, for instance from 0.5 to 5 mg/kg.
  • Suitable unit dosages will include from 10 mg to 400 mg, more preferably from 30 mg to 200 mg, e.g. from 40 mg to 160 mg.
  • Diphyllin is a plant-derived lignan, which previously has been investigated for cytotoxicity and anti-inflammatory effects ' ⁇ , however the mode of action behind these effects has until now been unknown ' . Moreover, diphyllin has been shown to have an antiproliferative activity towards human monocytes, with an effect that resulted in the cell cycle stopping in S-phase and inhibition of protein synthesis 24 , although this effect was obtain with 10,000 higher concentrations that the herein found specific effects on the V-ATPase.
  • AIA adjuvant induced arthritis
  • Diphyllin has been found to attribute to the toxicity of the plant Cleistanthus collinus together with its glycosides cleistanthin A, cleistanthin B and collinusin 35 .
  • An effect of the plant Cleistanthus collinus has been recorded involving glutathione depletion and adenosine triphosphatase (Na + , K + -ATPase, and Mg-ATPase) inhibition in liver, kidney, heart, brain and skeletal muscles 35 .
  • the current finding of activity as a potent V-ATPase inhibitor reveals the suitability of diphyllin as a treatment for different disease areas, e.g. osteoporosis and gastrointestinal disorders.
  • the V-ATPase is essential for osteoclastic bone resorption, and diphyllin and the other compounds of the invention may be used to treat postmenopausal osteoporosis.
  • Osteoclast acidification inhibitors have previously been shown to inhibit bone resorption without affecting bone formation 36 ' 37 .
  • this inhibitor opens new opportunities for treatment of osteoporosis.
  • Figure 1 shows the formula of diphyllin, (b) shows the formula of a typical glycoside, ramontoside, and (c) shows the formula of a further glycoside, cleisthanin A;
  • Figure 2 panels A and B, shows results obtained for diphyllin and a control compound bafilomycin respectively in Example 1 ;
  • FIG. 3 panels A and B, shows results obtained for diphyllin and a control compound bafilomycin respectively in Example 2 ;
  • Figure 4 panels A, B and C, shows (panel A) fluorescence photomicrographs of osteoclasts obtained in Example 3 and in panels B and C results obtained for diphyllin and a control compound bafilomycin respectively in an osteoclast acidification assay in Example 3.
  • Figure 5 shows in panels A, B and C the results obtained for diphyllin in Example 4 in a CTX release assay, a cell viability assay and a TRACP activity assay respectively; and
  • Figure 6 shows results for diphyllin obtained in Example 5 in a Ca 2+ release assay.
  • Figure 7, panel A and B, shows (panel A) fluorescence photomicrographs of HEK 293 cells treated with diphyllin obtained in Example 6 and panel B shows the result obtained for the reading of Acridine orange in the HEK 293 cells treated with diphyllin.
  • the acidification assay is described in Example 6.
  • Figure 8 shows results corresponding to those in Figure 3 but obtained for diphyllin analogues in Example 7.
  • Figure 9 shows results corresponding to those shown in Figures 4 and 5, but obtained for a first diphyllin analogue in Example 7.
  • Figure 10 shows results corresponding to those shown in Figures 4 and 5, but obtained for a second diphyllin analogue in Example 7.
  • Figure 11 shows results corresponding to those shown in Figure 2, but obtained for a first diphyllin analogue in example 7.
  • Figure 12 shows results corresponding to those shown in Figure 2, but obtained for a first diphyllin analogue in example 7.
  • Diphyllin used in this study was synthetic and was obtained from Asinex (BAS 00442101) .
  • Bafilomycin Al was purchased from Calbiochem.
  • BCG Bovine Chromaffin Granules
  • Bovine Chromaffin Granules were isolated from the medullae of bovine adrenal glands according to a previously published protocol 41 .
  • the medullary section of adrenal glands was cut free, the medullae were homogenized and ultra- centrifuged on a two-layer sucrose gradient, and the subsequent pellet was re-suspended, stored at -80°C and then used for both a V-ATPase assay and influx measurements.
  • V-ATPase activity was assayed using a modification of a previously published protocol 42 .
  • BCG were incubated in assay buffer (25 mM Tris-HCl, 50 mM KCl, 2.5 mM MgSO 4 , 0.5 mM EGTA, 3 mM DTT, 3 mM ATP, 2 mM ouabain, 3 mM Na-azide) and to eliminate contribution from P-ATPases and F-ATPases sodium vanadate (0.5 mM) and oligomycin (5 ⁇ g/ml) were included, and then diphyllin or bafilomycin was added. The reaction was performed at 37°C for 1 hour and was stopped by placing the plate on ice.
  • assay buffer 25 mM Tris-HCl, 50 mM KCl, 2.5 mM MgSO 4 , 0.5 mM EGTA, 3 mM DTT, 3 mM ATP, 2 mM ouabain, 3 mM Na-azide
  • the acid influx assay was performed as previously described 7 . Briefly, BCG was incubated in reaction buffer (5 mM Hepes, 150 mM KCl, 5 mM MgSO 4 , 1.5 ⁇ M Acridine orange, 1 nM valinomycin) , and the inhibitors diphyllin or bafilomycin were added. The reaction mixture was incubated at room temperature for 30 minutes. Reaction was initiated by addition of 5 mM ATP to the wells, and immediately after the plate was read in a plate reader using excitation 485 nm and emission 535 nm. The fluorescence was read every 15 seconds, and the results are presented in Figures 3A and 3B.
  • the osteoclasts ability to acidify the resorption lacuna is important for its function 43 .
  • acridine orange dye to investigate the response of human osteoclasts to diphyllin.
  • Acridine orange emits a bright orange fluorescence at a pH between 4 and 5 as observed in the resorption lacuna 44 .
  • osteoclasts in the absence of inhibitors have an orange fluorescence indicating the formation of a resorption lacuna.
  • Addition of diphyllin led to complete abrogation of the orange signal and the osteoclast acidification has been blocked totally at 500 nM. Diphyllin inhibits the acidification dose dependently.
  • the known V-ATPase inhibitor, bafilomycin Al (200 nM) was used as a positive control showing the same as diphyllin as expected 14 ' 36 .
  • the acidification was also measured qualitative by measurement of the fluorescence of the green signal (inhibited acidification) . This was done in the SpectraMax Gemini XS done at excitation 492 nm and emission 535 nm (green wavelength) . As seen in figure 4B both bafilomycin and diphyllin inhibits the acidification dose dependently resulting in a higher intensity of the fluorescence at the green wavelength.
  • Human CD14+ monocytes were isolated as previously described 45"47 . Briefly, the monocytes were isolated from peripheral blood by centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia) , and magnetically sorted using a CD14+ magnetic bead isolation kit (Dynabeads M-450, Dynal Biotech).
  • the cells were either seeded in 75cm 2 flasks for resorption or in 96 well plates for acidification experiments, and cultured in CCMEM containing 10% serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 25 ng/ml of M-CSF (R&D Systems) and 25 ng/ml RANKL (R&D Systems) for 10-12 days until mature osteoclasts were present.
  • CCMEM containing 10% serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 25 ng/ml of M-CSF (R&D Systems) and 25 ng/ml RANKL (R&D Systems) for 10-12 days until mature osteoclasts were present.
  • bafilomycin as a positive control inhibits the acidification totally at 200 nM.
  • Diphyllin panel A, inhibits acidification dose dependently and totally at 500 nM.
  • bafilomycin inhibits the acidification at 100 nM seen as an increase in the green fluorescence with acridine orange and in panel B diphyllin inhibits the acidification dose dependently from 125 nM.
  • Cortical bone slices were cut from sticks (Nordic Bioscience Diagnostics) , which were made by drilling sticks out of the cortical midshafts from bovine bone. The sticks were cut into small slices with a thickness of 0.2 mm with a diameter that fits into 96 well plates.
  • Diphyllin was added to the osteoclasts in different concentrations (1 ⁇ M, 0.3 ⁇ M, 0.1 ⁇ M, 30 nM, 10 nM, 3 nM and 1 nM) , while DMSO was added as vehicle and bafilomycin as a positive control (10 nM) .
  • DMSO was added as vehicle and bafilomycin as a positive control (10 nM) .
  • the cell viability was assessed by the AlamarBlue assay (figure 5B) and the release of the C-terminal type I collagen (CTX-I) and the TRACP activity was measured in the culture supernatant ( Figure 5A and 5C) .
  • CX-I c-terminal type I collagen fragments
  • Diphyllin inhibited calcium release from the bone slices during osteoclastic bone resorption, with an IC50 value of 49nM.
  • acridine orange dye to investigate the response of kidney cell (HEK 293 cells) to diphyllin.
  • Acridine orange emits a bright orange fluorescence at a pH between 4 and 5 due to acidification.
  • HEK cells in the absence of inhibitors have an orange fluorescence, while addition of diphyllin led to complete abrogation of the orange signal at 1 ⁇ M.
  • Diphyllin inhibits the acidification dose dependently.
  • the known V-ATPase inhibitor, bafilomycin Al (200 nM) was used as a positive control showing the same as diphyllin, as expected 14 ' 36 .
  • the acidification was also measured quantitatively by measurement of the fluorescence of the green signal (inhibited acidification) .
  • diphyllin inhibits the acidification dose dependently resulting in a higher fluorescence of the green wavelength.
  • the HEK 293 cells was used, and the cells were seeded in 96 well plates (50,000 cells/well) and cultured for 1 day.
  • Acridine orange (3, 6- bis [Dimethylamine] acridine) at 10 ⁇ g/ml was loaded for 45 min in the culture medium of osteoclasts in the presence or absence of bafilomycin or diphyllin as inhibitors.
  • the dye was washed and pictures were taken using an OLYMPUS IX-70 microscope and an OLYMPUS U-MWB filter (2OX objective) or the fluorescence were measured using the SpectraMax Gemini XS at excitation 492 nm and emission 535 nm.
  • R a is acetyl (compound PB24483) and where R a is ethoxy
  • Example 1 are shown in Figure 8.
  • Figure 8A shows the result for the diphyllin acetyl ester
  • Figure 8B shows the result for ethoxydiphyllin.
  • Figure 9 shows Figure 9 (PB24483) and Figure 10 (PB24485) .
  • the diphyllin acetyl ester and ethoxydiphyllin both inhibit the lysosomal acidification in osteoclasts ( Figure 9A and Figure 10A) . Furthermore, the diphyllin acetyl ester and ethoxydiphyllin inhibited bone resorption with IC50 values at 19nM and 8nM, respectively ( Figure 9B and Figure 10B) , without negative effects on osteoclast cell viability (Figure 9C and Figure 10C) . Interestingly, cell viability was significantly increased compared to control, indicating increased survival of osteoclasts 36 when resorption is inhibited like with diphyllin . The results for inhibition of the acid influx as per
  • Example 1 are shown in Figure 11 (PB24483) and Figure 12
  • Yoshimori, T . Yamamoto,A., Moriyama,Y., Futai,M. & Tashiro,Y. Bafilomycin Al, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266, 17707-17712 (1991).
  • Gozler,T. & Gozler,B. Konyanin A new lignan from Haplophyllum vulcanicum. Tetrahedron 40, 1145-1150 (1984) .

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Abstract

A V-ATPase or gastric proton pump inhibiting lignan such as diphyllin and related glycosides and derivatives, for use as a medicament in the treatment of excessive osteoclast action or in the treatment of excessive gastric acidification, suitably of the general formula (I), wherein : Rb and Rc may together form an alkylene bridge; Rd and Re also may together form an alkylene bridge; and each dotted bond independently is present or absent; and wherein the lactone ring is optionally opened and is optionally esterified.

Description

Diphyllin and other lignans as a medicament for V-ATPase mediated disease
The present invention relates to a newly discovered activity of lignans including the compound diphyllin in proton pump blocking for use for instance in treating gastric disorders or for reducing the activity of osteoclasts.
Proton pump inhibitors (PPI' s) such as omeprazole are a widely used class of pharmaceutical whose main action is pronounced and long-lasting reduction of gastric acid production. They have proved useful in the treatment of a range of conditions. They act by irreversibly blocking the hydrogen/potassium adenosine triphosphatase enzyme system (the H+/K+ ATPase, or more commonly just λgastric proton pump') of the gastric parietal cell. Cross-reactivity has previously been observed between V-ATPase (see below) blockers and gastric proton pump blockers1. The proton pump is the terminal stage in gastric acid secretion, being directly responsible for secreting H+ ions into the gastric lumen, making it an ideal target for inhibiting acid secretion .
Targeting the terminal-step in acid production, as well as the irreversible nature of the inhibition, results in a class of drugs that is significantly more effective than H2 antagonists and reduces gastric acid secretion by up to 99%.
The lack of the acid in the stomach will aid in the healing of duodenal ulcers, and reduces the pain from indigestion and heartburn, which is caused by excess stomach acid. Osteoclastic bone resorption is essential for maintenance of the skeleton. The resorption of the mineralized bone matrix mediated by osteoclasts occurs through active secretion of protons through a V-ATPase2 5, which is followed by passive transport of chloride ions through the chloride channel or exchanger ClC-7, thereby maintaining electroneutrality6"9. Vacuolar H+-ATPases (V-ATPases) are a class of proton pumps driven by ATP. This a sub-class of the multi-subunit proton pumps found in all eukaryotic cells, including osteoclasts10. The osteoclast V-ATPase contains an osteoclast specific subunit, the a3 subunit11'12. Loss of function mutations in the a3 subunit lead to severe osteopetrosis in both humans and mice, due to reduced acidification of the resorption lacuna3'5'13.
Few natural and some synthetic V-ATPase inhibitors are known, such as the natural compounds Bafilomycin Al14'15 and Concanamycin15 as well as the synthetic inhibitors FR16735616 and SB24278417. All these compounds potently inhibit osteoclastic resorption via inhibition of the acidification of the resorption lacuna, although the natural compounds are more than 100 fold more potent than their synthetic counterparts14"17.
Diphyllin (l-hydroxy-2- (hydroxymethyl) -6, 7-dimethoxy-4- (3, 4-methylenedioxy-phenyl) -3-naphthoic acid-γ-lactone) was initially isolated as a natural compound found so far in five different plants Cleistanthus collinus, Justicia procumbens, Haplophyllum bucharicum, Justicia patentiflora and Phyllanthus polyphyllus18'30.
Diphyllin is a member of a family of lignan natural products also called phytochemicals, and diphyllin is a
I Q I Q 00 — 99 r) 9 P, ^ D phenolic lignan lactone ' ' ' ' ' . There are many closely related lignans including for instance the justicidins and the taiwanins and still more somewhat less closely related lignans including the podophyllotoxins . Different glycosylated forms of the lignans also exist e.g. the Ramontosides and Cleistanthins19'23'25'28'30"33. The chemical structure of diphyllin is shown in Figure Ia. Ramontoside is diphyllin-4-O-glucopyranosyl (1-4) -2, 3-di-0- methylxylopyranoside . Its structure is shown in Figure Ib. Cleisthanin A is shown in Figure Ic.
The lignan natural products and their associated glycosides are related with a range of biological activities, which include cytotoxic effects as well as antitumor activity and anti-inflammatory effects18'23"25'27'28'33'34.
We have identified and characterised diphyllin as a novel and very potent inhibitor of the V-ATPase and as a potential inhibitor of the gastric proton pump. Accordingly, the present invention now provides a V-ATPase or gastric proton pump inhibiting lignan (or alternatively the use of a V-ATPase or gastric proton pump inhibiting lignan in the preparation of a medicament) for use in reducing the bone resorption activity of osteoclasts or in reducing gastric acidification, thus being useful in the treatment of excessive osteoclast action (e.g. loss of bone mass) or in the treatment of excess gastric acidification.
Said lignan is preferably of the general formula 1
Figure imgf000005_0001
Formula 1
wherein : Ra is:
-H; a glycosylating moiety; an amino glycosylating moiety; alkyl, preferably Ci-6, alkaryl, in which the alkyl moiety is preferably Ci-6, aralkyl, in which the alkyl moiety is preferably Ci-6, e.g. benzyl, cycloalkyl, preferably C3-6, alkenyl, preferably C2-6 , carboxyalkyl in which the alkyl moiety is preferably Ci-6, alkoxycarbonyl, e.g. C1-4 alkoxycarbonyl, alkoxycarbonylalkyl, e.g. Ci-4alkoxycarbonyl-Ci-4alkyl, alkylcarbonyl, e.g. Ci- 4alkylcarbonyl, alkylcarbonylalkyl, e.g. C1-4 alkylcarbonyl-Ci-4alkyl, carboxy, or ORa, wherein Ra is as defined above, or alkylcarbonyloxy, e.g. C1-4 alkylcarbonyloxy;
Rb and Rc each independently is a group listed above in relation to Ra, or together form an alkylene bridge, preferably a methylene bridge; Rd and Re each independently is a group listed above in relation to Ra, or together form an alkylene bridge, preferably a methylene bridge;
Rf independently is a group listed above in relation to Ra, or ORf is replaced by H;
Rg is H or is F, -CF3, -CH3, -00CH3, -ORh, or -SRh, where Rh is H, or alkyl;
R1 independently is a group listed above in relation to Ra, for instance -OH, -OMe, or -Me; each dotted bond independently is present or absent; and wherein the lactone ring is optionally opened and is optionally esterified at the resulting hydroxyl and/or the resulting carboxylic acid functionality, but wherein Rb and Rc do not together form an alkylene bridge except when (a) Rd and Re together form an alkylene bridge, or (b) wherein the dotted bonds are all present, or (c) wherein both (a) and (b) apply.
Said lignan may be of the general formula 2
Figure imgf000006_0001
Formula 2 wherein Ra~f are as defined above and each dotted bond is independently present or absent,
The lignan may be of the general formula 3
Figure imgf000007_0001
Formula 3 wherein Ra~e are as defined above and each dotted bond is independently present or absent. In each of Formulae 1-3 it is preferred that Rd and Re form an alkylene, preferably methylene, bridge and in Formulae 1 and 2 that Rf is H. Also, separately or in combination with this, it is preferred that one or both of the dotted double bonds is present as a double bond.
The lignan may be of the general formula 4:
Figure imgf000008_0001
Formula 4
wherein Ra c are as defined above.
Rb and Rc are each preferably methyl, and Ra is preferably hydroxyl or a glycoside group.
The active compound may be according to the general formula 5
Figure imgf000008_0002
Formula 5 wherein Ra is :
-H (justicidin B) a glycosylating moiety an amino glycosylating moiety alkyl (e.g. Ci-6 alkyl, especially methyl), alkaryl, e.g. Ci-6 alkylaryl, aralkyl, e.g. aryl Ci-6 alkyl (in each of which aryl may be phenyl), cycloalkyl, e.g. C3-6 cycloalkyl, alkenyl, e.g. C2-6 alkenyl, carboxyalkyl, e.g. carboxy Ci-6 alkyl, or alkylcarbonyloxy, e.g. C1-4 alkylcarbonyloxy, such as acetyl, or
ORa, wherein Ra is as defined above, e.g. is H (diphyllin) , or methyl (Justicidin A), or ethyl.
Suitable glycosylating moieties include β-D- apiofuranosyloxy, β-D-xylopyranosyl- (1→2) -β-D- apiofuranosyloxy, and α-L-arabinopyranosyl- (1→6) -β-D- glucopyranosyloxy, or the glcosylation seen in patavine 34.
Suitable arylnaphthalene lignans include diphyllin, ethoxydiphyllin and other alkoxydiphyllins, acetyldiphyllin and other diphyllin esters, justicidin (A or B) , patavine, and cleisthanins (including A and B) .
In formula 5, where Ra is as shown below, the resulting compounds are as follows:
Figure imgf000010_0003
However, the podophyllotoxins :
Figure imgf000010_0001
Figure imgf000010_0002
have been found not to be active in the invention.
Examples of compounds for use in the invention include the following compounds tested in Example 8:
Figure imgf000011_0001
PB24483 PB24485
The invention includes these compounds per se.
Synthesis of compounds of this family, including diphyllin itself is described in for instance GB-A-1178341.
The invention includes methods of treatment of V-ATPase or gastric proton pump mediated diseases comprising administering to a patient in need thereof a therapeutically effective amount of an active compound described herein. Active compounds of the invention may be used in the treatment of excess gastric acidification or excess gastric acidification diseases including peptic ulcer disease, duodenal ulcers, reflux esophagitis, heartburn, Helicobacter pylori positive duodenal ulcer disease, Helicobacter pylori positive gastric ulcer disease, gastroduodenal ulceration associated with the use of non-steroidal anti-inflammatory drugs (NSAIDs) , acid-related indigestion (dyspepsia) , aspiration pneumonitis and rare illnesses such as Zollinger- Ellison syndrome, as well as inflammatory bowel diseases, such as Morbus Crohn (Crohn's disease) and Colitis Ulcerosa (ulcerative colitis) , and also in osteoclast mediated diseases, particularly those diseases in which osteoclast activity in resorbing bone is excessive, including excessive bone resorption, bone mass loss, osteoporosis, hyperparathyroidism, Paget ' s disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilisation or sex hormone deficiency, or osteomalacia. Bone loss can also be a symptom associated with Crohn's disease and ulcerative colitis, or with the treatment of these and other conditions with corticosteroids, and can be treated according to the invention. Treatment according to the invention may be for the relief of symptoms or may be preventative and administered in advance of symptoms appearing.
For administration for the treatment of such conditions, compounds of the invention, including diphyllin, may be administered orally or in some instances rectally, and may be formulated as tablets or capsules, optionally in enteric forms, including enterically coated granules within a capsule and multi- pellet unit forms, and/or slow release forms. Alternative forms include chewable tablets and powders. The compounds may also be formulated for administration parentally, including injectable forms for e.g. IV administration. Alternatively, the compounds of the invention, especially diphyllin or its glycosides, may be incorporated in a nutraceutical product which may take the form of a solid foodstuff such as a cereal bar or a liquid or infusible product for making a drink, possibly a hot drink such as a λtea' .
The invention therefore includes a pharmaceutical or veterinary composition comprising an active compound of the invention mixed with a pharmaceutically or veterinarily acceptable carrier or excipient material, said composition preferably being a solid composition.
Suitable dosage levels of the active will be from 0.01 to 1000 mg/kg, more preferably 0.1 to 100 mg/kg, for instance from 0.5 to 5 mg/kg. Suitable unit dosages will include from 10 mg to 400 mg, more preferably from 30 mg to 200 mg, e.g. from 40 mg to 160 mg.
By random screening of compounds for their ability to inhibit acidification in an influx assay, we first identified the natural compound diphyllin. We found that diphyllin was a very potent inhibitor of the H+-V-ATPase, and that it via inhibition of the H+-V-ATPase had a strong inhibitory effect on vesicular acidification and as a consequence to that osteoclastic acidification and resorption. These data are the first to associate a precise molecular mode of action of diphyllin, in the pico-molar range, even though several effects have been suggested although in the micro molar range. Even though diphyllin long had been tested for pharmacological affects in other biological systems, to date, no precise molecular mode of action has been presented.
Diphyllin is a plant-derived lignan, which previously has been investigated for cytotoxicity and anti-inflammatory effects ' ~ , however the mode of action behind these effects has until now been unknown ' . Moreover, diphyllin has been shown to have an antiproliferative activity towards human monocytes, with an effect that resulted in the cell cycle stopping in S-phase and inhibition of protein synthesis24, although this effect was obtain with 10,000 higher concentrations that the herein found specific effects on the V-ATPase. Evidence for an anti inflammatory effect of V-ATPase inhibitors, although with synthetic compounds with significantly lower potencies, is found in adjuvant induced arthritis (AIA) models, in which both inflammation and bone destruction was inhibited41.
Diphyllin has been found to attribute to the toxicity of the plant Cleistanthus collinus together with its glycosides cleistanthin A, cleistanthin B and collinusin35. An effect of the plant Cleistanthus collinus has been recorded involving glutathione depletion and adenosine triphosphatase (Na+, K+-ATPase, and Mg-ATPase) inhibition in liver, kidney, heart, brain and skeletal muscles35.
The current finding of activity as a potent V-ATPase inhibitor reveals the suitability of diphyllin as a treatment for different disease areas, e.g. osteoporosis and gastrointestinal disorders.
In bone, the V-ATPase is essential for osteoclastic bone resorption, and diphyllin and the other compounds of the invention may be used to treat postmenopausal osteoporosis. Osteoclast acidification inhibitors have previously been shown to inhibit bone resorption without affecting bone formation36'37. In contrast to all other anti-resorptive treatments that secondarily inhibit bone resorption38 due to the coupling principle39'40, this inhibitor opens new opportunities for treatment of osteoporosis.
With respect to gastro-intestinal disorders synthetic proton pump inhibitors have long been used for e.g. gastroesophageal reflux disease, stomach ulcers, acid-related indigestion (dyspepsia) , and rare illnesses such as Zollinger-Ellison syndrome, a disease associated with ulcers and severely increased production of acid in the stomach. Thus, this novel inhibitor family, especially diphyllin itself, due to its potency is able to benefit many patient groups .
The present invention will be further described and illustrated with reference to the accompanying drawings in which:
Figure 1 (a) shows the formula of diphyllin, (b) shows the formula of a typical glycoside, ramontoside, and (c) shows the formula of a further glycoside, cleisthanin A; Figure 2, panels A and B, shows results obtained for diphyllin and a control compound bafilomycin respectively in Example 1 ;
Figure 3 panels A and B, shows results obtained for diphyllin and a control compound bafilomycin respectively in Example 2 ; Figure 4, panels A, B and C, shows (panel A) fluorescence photomicrographs of osteoclasts obtained in Example 3 and in panels B and C results obtained for diphyllin and a control compound bafilomycin respectively in an osteoclast acidification assay in Example 3. Figure 5 shows in panels A, B and C the results obtained for diphyllin in Example 4 in a CTX release assay, a cell viability assay and a TRACP activity assay respectively; and
Figure 6 shows results for diphyllin obtained in Example 5 in a Ca2+ release assay. Figure 7, panel A and B, shows (panel A) fluorescence photomicrographs of HEK 293 cells treated with diphyllin obtained in Example 6 and panel B shows the result obtained for the reading of Acridine orange in the HEK 293 cells treated with diphyllin. The acidification assay is described in Example 6.
Figure 8 shows results corresponding to those in Figure 3 but obtained for diphyllin analogues in Example 7.
Figure 9 shows results corresponding to those shown in Figures 4 and 5, but obtained for a first diphyllin analogue in Example 7.
Figure 10 shows results corresponding to those shown in Figures 4 and 5, but obtained for a second diphyllin analogue in Example 7.
Figure 11 shows results corresponding to those shown in Figure 2, but obtained for a first diphyllin analogue in example 7. Figure 12 shows results corresponding to those shown in Figure 2, but obtained for a first diphyllin analogue in example 7.
The following examples demonstrate the efficacy of diphyllin and analogues in modulating the activity of osteoclasts .
In these examples, Statistical analyses were performed using one-way analysis of variance followed by Dunnett's multiple comparison tests. Bartlett's test was used to assess variance homogeneity. Statistical significance is indicated by the number of asterisks, p<0.05* and p<0.001**'1
Diphyllin used in this study was synthetic and was obtained from Asinex (BAS 00442101) . Bafilomycin Al was purchased from Calbiochem.
Examples Example 1
Inhibition of acid influx via V-ATPase by diphyllin
To investigate whether diphyllin inhibited acid influx, we used Bovine Chromaffin Granules (BCG) in an acid influx assay. We compared diphyllin to the known H+-V-ATPase inhibitor bafilomycin Al, which potently inhibits acidification in this assay with an IC50 value of 0.4 nM (Figure 2A) . We found that diphyllin inhibited acidification in the influx assay potently (Figure 2B) with an IC50 of 0.6 nM. Thus, showing that diphyllin is very potent for inhibition of acidification.
Bovine Chromaffin Granules (BCG) were isolated from the medullae of bovine adrenal glands according to a previously published protocol41. The medullary section of adrenal glands was cut free, the medullae were homogenized and ultra- centrifuged on a two-layer sucrose gradient, and the subsequent pellet was re-suspended, stored at -80°C and then used for both a V-ATPase assay and influx measurements. V-ATPase activity was assayed using a modification of a previously published protocol42. Briefly, BCG were incubated in assay buffer (25 mM Tris-HCl, 50 mM KCl, 2.5 mM MgSO4, 0.5 mM EGTA, 3 mM DTT, 3 mM ATP, 2 mM ouabain, 3 mM Na-azide) and to eliminate contribution from P-ATPases and F-ATPases sodium vanadate (0.5 mM) and oligomycin (5 μg/ml) were included, and then diphyllin or bafilomycin was added. The reaction was performed at 37°C for 1 hour and was stopped by placing the plate on ice. The formation of free phosphate was measured by addition of molybdate reagent (1.8 mM ammonium molybdate, 270 mM H2SO4, 1% (w/v) ascorbic acid) . Finally, the results were quantified by measuring absorbance at 750 nM. As background, samples incubated on ice were used, and these results were subtracted from the values obtained at 37°C.
Example 2 Inhibition of V-ATPase activity by diphyllin
Since acid influx to the resorption lacunae of osteoclasts is dependent on both proton and chloride transport , we investigated whether diphyllin showed inhibitory activity in an H+-V-ATPase assay. The BCG fraction has previously been shown to contain high levels of H+-V-ATPase activity41, and using the known H+-V-ATPase inhibitor bafilomycin we found that it potently inhibited H+- V-ATPase activity with an IC50 of 4 nM (Figure 3A) . We then investigated the effect of diphyllin, and found that it also inhibited H+-V-ATPase activity (Figure 3B) with an IC50 value of 17 nM.
The acid influx assay was performed as previously described7. Briefly, BCG was incubated in reaction buffer (5 mM Hepes, 150 mM KCl, 5 mM MgSO4, 1.5 μM Acridine orange, 1 nM valinomycin) , and the inhibitors diphyllin or bafilomycin were added. The reaction mixture was incubated at room temperature for 30 minutes. Reaction was initiated by addition of 5 mM ATP to the wells, and immediately after the plate was read in a plate reader using excitation 485 nm and emission 535 nm. The fluorescence was read every 15 seconds, and the results are presented in Figures 3A and 3B. The effects of the inhibitors are quantified as the slope of the acidification influx in all conditions, and they are presented in percent of the vehicle condition. To ensure that the compounds had no effect on the fluorescence the plate was read before addition of ATP to obtain a steady state level. Example 3
Inhibition of osteoclast acidification by diphyllin
The osteoclasts ability to acidify the resorption lacuna is important for its function43. We used acridine orange dye to investigate the response of human osteoclasts to diphyllin. Acridine orange emits a bright orange fluorescence at a pH between 4 and 5 as observed in the resorption lacuna44. As seen in figure 4A, osteoclasts in the absence of inhibitors have an orange fluorescence indicating the formation of a resorption lacuna. Addition of diphyllin led to complete abrogation of the orange signal and the osteoclast acidification has been blocked totally at 500 nM. Diphyllin inhibits the acidification dose dependently. The known V-ATPase inhibitor, bafilomycin Al (200 nM) , was used as a positive control showing the same as diphyllin as expected14'36. The acidification was also measured qualitative by measurement of the fluorescence of the green signal (inhibited acidification) . This was done in the SpectraMax Gemini XS done at excitation 492 nm and emission 535 nm (green wavelength) . As seen in figure 4B both bafilomycin and diphyllin inhibits the acidification dose dependently resulting in a higher intensity of the fluorescence at the green wavelength.
Human CD14+ monocytes were isolated as previously described45"47. Briefly, the monocytes were isolated from peripheral blood by centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia) , and magnetically sorted using a CD14+ magnetic bead isolation kit (Dynabeads M-450, Dynal Biotech). The cells were either seeded in 75cm2 flasks for resorption or in 96 well plates for acidification experiments, and cultured in CCMEM containing 10% serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 ng/ml of M-CSF (R&D Systems) and 25 ng/ml RANKL (R&D Systems) for 10-12 days until mature osteoclasts were present.
To perform the osteoclast acidification assay Acridine orange (3, 6-bis [Dimethylamine] acridine) at 10 μg/ml was loaded for 45 min in the culture medium of osteoclasts in the presence or absence of bafilomycin or diphyllin as inhibitors. The dye was washed and pictures were taken using an OLYMPUS IX-70 microscope and an OLYMPUS U-MWB filter (2OX objective) or the fluorescence were measured using the SpectraMax Gemini XS at excitation 492 nm and emission 535 nm.
As seen in Figure 4, panel A, bafilomycin as a positive control inhibits the acidification totally at 200 nM. Diphyllin, panel A, inhibits acidification dose dependently and totally at 500 nM. In panel C, bafilomycin inhibits the acidification at 100 nM seen as an increase in the green fluorescence with acridine orange and in panel B diphyllin inhibits the acidification dose dependently from 125 nM.
Example 4
Inhibition of osteoclastic resorption of bone by diphyllin
To investigate the effect of diphyllin on human osteoclastic bone resorption, mature human osteoclasts were lifted and seeded on cortical bovine bone slices in the presence or absence of diphyllin for the bone resorption experiments, and allowed to attach and initiate bone resorption for one day, as previously described45'48'49. Cortical bone slices were cut from sticks (Nordic Bioscience Diagnostics) , which were made by drilling sticks out of the cortical midshafts from bovine bone. The sticks were cut into small slices with a thickness of 0.2 mm with a diameter that fits into 96 well plates. Diphyllin was added to the osteoclasts in different concentrations (1 μM, 0.3 μM, 0.1 μM, 30 nM, 10 nM, 3 nM and 1 nM) , while DMSO was added as vehicle and bafilomycin as a positive control (10 nM) . After five days of culture the cell viability was assessed by the AlamarBlue assay (figure 5B) and the release of the C-terminal type I collagen (CTX-I) and the TRACP activity was measured in the culture supernatant (Figure 5A and 5C) .
The release of c-terminal type I collagen fragments (CTX-I) was determined using the CrossLaps for Culture kit (Nordic Bioscience Diagnostics) according to the manufacturer's instructions.
For each assay, the results are presented as relative inhibition compared to vehicle treated osteoclasts. Diphyllin inhibited bone resorption with an IC50 value at 14 nM, without negative effects on osteoclast cell viability. Interestingly, cell viability was significantly increased compared to control, indicating increased survival of osteoclasts36 when resorption is inhibited. We measured TRACP activity, which in line was increased when the resorption was inhibited. This is also observed with the known V-ATPase inhibitor bafilomycin36.
Example 5 Inhibition of calcium release during osteoclastic bone resorption with diphyllin
Dissolution of the inorganic phase of bone is a prerequisite for bone resorption, and results in release of the calcium from bone. As diphyllin highly potently inhibited acidification and bone resorption, we characterized the human osteoclastic resorption by assessing the calcium release from the bone slices. Mature human osteoclasts were seeded on cortical bovine bone slices, and allowed to attach and initiate bone resorption for one day. Diphyllin was added to the osteoclasts in different concentrations (1 μM, 0.3 μM, 0.1 μM, 30 nM, 10 nM, 3 nM and 1 nM) , while DMSO was added as vehicle and bafilomycin as a positive control. After five days of culture the release of calcium was measured using the Hitachi 912 Automatic Analyzer (Boehringer Mannheim) (Figure 6) .
Diphyllin inhibited calcium release from the bone slices during osteoclastic bone resorption, with an IC50 value of 49nM.
Example 6
Inhibition of acidification in HEK 293 cells by diphyllin
We used acridine orange dye to investigate the response of kidney cell (HEK 293 cells) to diphyllin. Acridine orange emits a bright orange fluorescence at a pH between 4 and 5 due to acidification. As seen in figure 7A, HEK cells in the absence of inhibitors have an orange fluorescence, while addition of diphyllin led to complete abrogation of the orange signal at 1 μM. Diphyllin inhibits the acidification dose dependently. The known V-ATPase inhibitor, bafilomycin Al (200 nM) , was used as a positive control showing the same as diphyllin, as expected14'36. The acidification was also measured quantitatively by measurement of the fluorescence of the green signal (inhibited acidification) . This was done in the SpectraMax Gemini XS done at excitation 492 nm and emission 535 nm (green wavelength) . As seen in figure 7B diphyllin inhibits the acidification dose dependently resulting in a higher fluorescence of the green wavelength. To perform the acidification assay, the HEK 293 cells was used, and the cells were seeded in 96 well plates (50,000 cells/well) and cultured for 1 day. Acridine orange (3, 6- bis [Dimethylamine] acridine) at 10 μg/ml was loaded for 45 min in the culture medium of osteoclasts in the presence or absence of bafilomycin or diphyllin as inhibitors. The dye was washed and pictures were taken using an OLYMPUS IX-70 microscope and an OLYMPUS U-MWB filter (2OX objective) or the fluorescence were measured using the SpectraMax Gemini XS at excitation 492 nm and emission 535 nm.
As seen in Figure 7, panel A and B, Bafilomycin, as a positive control, inhibits the acidification totally at 200 nM. In panel A, Diphyllin inhibits acidification dose dependently and totally at 1 μM. In panel B, Diphyllin inhibits the acidification from 250 nM seen as an increase in the green fluorescence with acridine orange. Diphyllin inhibits acidification dose dependently.
Example 7
Inhibition of acid influx via the V-ATPase, osteoclast acidification and bone resorption by diphyllin acetyl ester and by ethoxydiphyllin
The assays of Examples 1, 2, 3 and 4 were repeated using varying concentrations of the derivative of diphyllin where
Ra is acetyl (compound PB24483) and where Ra is ethoxy
(compound PB24485) . Results for the V-ATPase assay (as per
Example 1) are shown in Figure 8. Figure 8A shows the result for the diphyllin acetyl ester, while Figure 8B shows the result for ethoxydiphyllin. We found that the two analogues inhibited H+-V-ATPase activity (Figure 8A and 8B) with IC50 values of 39 nM for both compounds. Results for the acidification assay as per Example 3 and for the CTX bone resorption assay as per Example 4, including cell viability by Alamar Blue, are shown in Figure 9 (PB24483) and Figure 10 (PB24485) . The diphyllin acetyl ester and ethoxydiphyllin both inhibit the lysosomal acidification in osteoclasts (Figure 9A and Figure 10A) . Furthermore, the diphyllin acetyl ester and ethoxydiphyllin inhibited bone resorption with IC50 values at 19nM and 8nM, respectively (Figure 9B and Figure 10B) , without negative effects on osteoclast cell viability (Figure 9C and Figure 10C) . Interestingly, cell viability was significantly increased compared to control, indicating increased survival of osteoclasts36 when resorption is inhibited like with diphyllin . The results for inhibition of the acid influx as per
Example 1 are shown in Figure 11 (PB24483) and Figure 12
(PB24485) . The diphyllin acetyl ester and ethoxydiphyllin both inhibit the acid influx in the osteoclast vesicles with
IC50 values at 1.5nM and 1.4nM, respectively. These results demonstrate that both compounds are powerful blockers of V-ATPase with IC50 values in the nM range, albeit not as powerful as diphyllin itself.
In this specification, unless expressly otherwise indicated, the word λor' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ^exclusive or' which requires that only one of the conditions is met. The word Comprising' is used in the sense of including' rather than in to mean Consisting of . All prior teachings acknowledged above are hereby incorporated by reference. Reference List
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Claims

A V-ATPase or gastric proton pump inhibiting lignan for use as a medicament in the treatment of excessive osteoclast action or in the treatment of excessive gastric acidification .
A lignan as claimed in claim 1, wherein said lignan is of the general formula 1 :
Figure imgf000031_0001
wherein :
Ra is:
-H; a glycosylating moiety; an amino glycosylating moiety; alkyl, alkaryl, aralkyl, cycloalkyl, alkenyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyl, alkylcarbonylalkyl, carboxy, or ORa, wherein Ra is as defined above, or alkylcarbonyloxy;
Rb and Rc each independently is: a group listed above in relation to Ra, or together form an alkylene bridge;
Rd and Re each independently is: a group listed above in relation to Ra, or together form an alkylene bridge; Rf independently is a group listed above in relation to
Ra, or 0Rf is replaced by H;
Rg is H or is F, -CF3, -CH3, -00CH3, -ORh, or -SRh, where
Rh is H, or alkyl;
R1 independently is a group listed above in relation to Ra, for instance -OH, -OMe, or -Me; each dotted bond independently is present or absent; and wherein the lactone ring is optionally opened and is optionally esterified, but (a) wherein Rb and Rc do not together form an alkylene bridge except when Rd and Re together form an alkylene bridge, or (b) wherein the dotted bonds are all present, or (c) wherein both (a) and (b) apply.
3. A lignan as claimed in claim 2, wherein Rb and Rc each independently is a group listed in claim 2 in relation to Ra.
4. A lignan as claimed in claim 2 or claim 3, wherein Rd and Re together form an alkylene bridge.
5. A lignan claimed in any one of claims 2 to 4, wherein said lignan is of the general formula 2:
Figure imgf000033_0001
wherein Ra f are as defined in any one of claims 1 to 4 and each dotted bond independently is present or absent.
6. A lignan as claimed in claim 4, wherein the lignan is of the general formula 3:
Figure imgf000033_0002
wherein Ra e are as defined in any one of claims 1 to 4, and each dotted bonds is independently present or absent .
7. A lignan as claimed in claim 4, wherein the lignan is of the general formula 4 :
Figure imgf000034_0001
wherein Ra c are as defined in claim 1
A lignan as claimed in claim 7, wherein said lignan is of the general formula 5:
Figure imgf000034_0002
Formula 5 wherein Ra is : -H, alkoxy, alkylcarbonyloxy, a glycosylating moiety, an amino glycosylating moiety or hydroxyl .
9. A lignan as claimed in claim 8, wherein Rb and Rc are each methyl, and Ra is hydroxyl, methoxy, ethoxy, methylcarbonyloxy, or a glycoside group.
10. A lignan as claimed in any preceding claim, wherein the use is in the treatment of peptic ulcer disease, duodenal ulcers, reflux esophagitis, heartburn, Helicobacter pylori positive duodenal ulcer disease, Helicobacter pylori positive gastric ulcer disease, gastroduodenal ulceration associated with the use of non-steroidal anti-inflammatory drugs (NSAIDs) , acid- related indigestion (dyspepsia) , aspiration pneumonitis, Crohn's disease, ulcerative colitis, or Zollinger- Ellison syndrome.
11. A lignan as claimed in any preceding claim, wherein the use is in the treatment of osteoporosis, hyperparathyroidism, Paget ' s disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilisation or sex hormone deficiency, or osteomalacia.
12. A nutraceutical product comprising a V-ATPase or gastric proton pump inhibiting lignan compound as defined in any preceding claim, optionally in combination with one or more nutritionally active materials.
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