Patent Grant
9751903
The present invention relates to enteric activators of Clostridium difficile toxin, particularly poly-phosphate derivatives, polysulfate derivatives or mixed polyphosphate/sulphate derivatives of six-membered cyclic polyols comprising a polyethylene glycol (PEG) moiety.
Clostridium difficile is a species of Gram-positive bacteria that causes severe diarrhoea in human patients. C. difficile infection (CDI) typically affects patients under antibiotic treatment since the bacterium is only able to colonize the colon of patients with depleted bacterial flora. The emergence of antibiotic-resistant strains of C. difficile causes increasingly severe morbidity and mortality due to the spread of new, more virulent strains, with recent outbreaks in North America and Europe.
C. difficile asymptomatically colonizes 2-5% of the human adult population. The bacteria form spores, which are difficult to neutralize by common methods of disinfection. As a result, C. difficile infections are a common result of prolonged stays in hospitals; the pathogen is considered the leading cause of hospital-associated diarrhoea in the USA.
Current therapy of choice is oral application of metronidazole or, in case of failure of the former, vancomycin. Since clinical symptoms of CDI are caused by two toxic proteins secreted by C. difficile in the colon, rather than by the presence of the bacteria itself, efforts have been made recently to target these toxins (e.g. employing polymeric binders), but have so far failed in clinical trials.
C. difficile enterotoxin (toxin A, TcdA) and cytotoxin (toxin B, TcdB) are the main contributors to the symptoms of disease (for a toxin biology review, see Voth and Ballard, Clinical Microbiology Reviews 2005, 18, 247-263). In brief, both toxins are composed of four domains, a first domain mediating the attachment of the toxin to cells; a second one facilitating translocation into the cytosol; a third domain causing the cleavage of the toxic domain by autoproteolysis, and finally the toxic domain or “warhead” itself, which causes the physiological effects of the toxin in the affected cell.
Reineke et al. (Nature 2007, 446, 415) identified myo-inositol hexakisphosphate (IP6) as the natural trigger of TcdA/TcdB autoprocessing in the cell cytosol. Egerer et al. (PLoS Pathog. 2010, 6, e1000942) and Shen et al. (Nat. Struct. Mol. Biol. 2011, 18, 364) suggested targeting the IP6-induced autoprocessing mechanism as a means of therapeutic intervention against toxin-mediated pathogenicity.
Kreimeyer et al. suggested using IP6 pharmaceutically to intervene in CDI (Naunyn-Schmiedeberg's Arch. Pharmacol. 2011, 383, 253). However, this approach is not feasible as the presence of high calcium concentrations in the colon precipitates IP6 and prevents it from being active.
Thus, the objective of the present invention is to provide improved treatment options for patients suffering from CDI. This objective is attained by the subject-matter of the independent claims.
The invention is based on a novel design of small-molecule analogues of IP6 that are provided as an oral therapy to trigger the cleavage of the toxin in the colon lumen, thereby detaching the toxin warhead before it reaches its destination, and rendering it harmless. IP6 itself cannot be used for this purpose because it is not soluble at the high calcium concentrations found in the colon lumen. The present invention provides novel IP6 analogues with improved solubility.
According to a first aspect of the invention, a pharmaceutical compound characterized by a general formula (1) is provided,
wherein
In certain embodiments, R1 is a polyethylene glycol.
In certain embodiments, R1 is a polyethylene glycol described by a formula (R3—(O—OCH2—CH2)n—) and R3 is hydrogen, methyl or ethyl, and n has a value from 2 to 200.
In certain embodiments, n has a value from 3 to 200.
In certain embodiments, n has a value from 3 to 20. In certain embodiments, n has a value from 10 to 30. In certain embodiments, n has a value from 9 to 45.
In certain embodiments, n has a value from 7 to 11.
In certain embodiments, n has a value of 2. In certain embodiments, n has a value of 7. In certain embodiments, n has a value of 9. In certain embodiments, n has a value of 11. In certain embodiments, n has a value of 45.
In certain embodiments, said polyethylene glycol is a branched polyethylene glycol.
In certain embodiments, R1 comprises an ether, thioether, carboxylic ester, amine, carboxylic amide, urea, sulfonamide, phosphoramide, phosphate ester, phosphorothioate, alkyl, triazole or carbamate function, or a combination of any of the preceding groups, which links R2 to the molecule.
In certain embodiments, R1 is a CH2 or a CH2CH2 group linking R2 to the molecule.
Formula (1) describes a six-membered alkyl ring and R1 is covalently attached to one of the carbon atoms (a CH moiety) forming the ring.
In certain embodiments, R2 is a polyglycerol described by the formula ((R3—O—(CH2—CHOH—CH2O)n—) with R3 being hydrogen, methyl or ethyl, and n having a value from 3 to 200. In some alternatives of these embodiments, n has a value from 3 to 20. In some alternatives of these embodiments, n has a value from 10 to 30. In some alternatives of these embodiments, n has a value from 9 to 45.
In certain embodiments, R2 is a branched polyglycerol described by the formula (R3—O—(CH2—CHOR5—CH2—O)n—) with R5 being hydrogen or a linear glycerol chain described by the formula (R3—O—(CH2—CHOH—CH2—O)n—) and R3 being hydrogen, methyl or ethyl.
In certain embodiments, R2 is a hyperbranched polyglycerol described by the formula (R3—O—(CH2—CHOR5—CH2—O)n—) with R5 being hydrogen or a glycerol chain described by the formula (R3—O—(CH2—CHOR6—CH2—O)n—), with R6 being hydrogen or a glycerol chain described by the formula (R3—O—(CH2—CHOR7—CH2—O)n—), with R7 being hydrogen or a linear glycerol chain described by the formula (R3—O—(CH2—CHOH—CH2—O)n—) and R3 being hydrogen, methyl or ethyl.
Hyperbranched glycerol and methods for its synthesis are described in Oudshorn et al., Biomaterials (2006), 27, 5471-5479; Wilms et al., Acc. Chem. Res. (2010) 43, 129-41, and references cited therein.
In embodiments wherein the solubility function is attached to the compound directly, R1 equals (is) R2.
The solubility function provides for the solubility of the molecule in aqueous solution in the presence of 10 mmol/l Ca2+. The molecule according to the invention has a higher solubility than IP6 in concentrations of calcium higher than 1 mmol/l. In certain embodiments, the solubility of the molecule of the invention is above 10 μmol/l.
The compound of the invention comprises a six-membered ring, wherein ring members can be described by a formula CH—X, and one ring member can be described by a formula CH—R1, wherein each X (independently of any other X) and R1 have the meaning defined above.
In certain embodiments, the compound according to the invention is characterized by a general formula (2a) or (3a)
wherein each X (independently) and R1 have the meaning outlined above.
In certain embodiments, the compound according to the invention is characterized by a general formula (2b) or (3b)
wherein each X (independently) and R1 have the meaning outlined above.
In certain embodiments, the compound of the invention is described by a formula (1). In certain embodiments, it is described by formulae (2a) or (2b). In certain embodiments, it is described by formulae (3a) or (3b).
In certain embodiments, the compound is described by a general formula (4a) or (4b)
wherein R1 has the same meaning as indicated above.
In one embodiment, (4a) comprises a poly(ethylene glycol) PEG400 as solubility function, with R1 being CH3(OCH2—CH2)9—O—:
myo-inositol-pentakisphosphate-2-PEG(400) (7) is the PEG400-analogue of myo-inositol hexakisphosphate. (7) has proved to have an improved ability to cleave TcdB CPD in the presence of calcium.
Another embodiment relates to the PEG2000 analogue to (7), i.e. a PEG with approximately 45 ethylene glycol monomers.
Another embodiment relates to the PEG100 analogue to (7), i.e. a PEG with 2 ethylene glycol monomers.
Another embodiment relates to the PEG320 analogue to (7), i.e. a PEG with 7 ethylene glycol monomers.
Another embodiment relates to the PEG500 analogue to (7), i.e. a PEG with 11 ethylene glycol monomers.
Another embodiment relates to an analogue to scyllo-inositol hexakisphosphate as described by formula (4b)
wherein R1 has the same meaning as outlined above. In some embodiments, the compound of the invention can be described by formula (4b) and R1 is a poly(ethylene glycol) moiety.
In certain embodiments, the compound is described by a general formula (5a) or (5b)
wherein R1 has the same meaning as indicated above.
In certain embodiments, the compound is described by a general formula (6a) or (6b)
wherein R1 has the same meaning as indicated above.
In certain embodiments, the polyethylene glycol is CH3(OCH2—CH2)9—O— or CH3(OCH2—CH2)45—O—.
In certain embodiments, the polyethylene glycol is CH3(OCH2—CH2)2—O—, CH3(OCH2—CH2)7—O— or CH3(OCH2—CH2)11—O—.
In certain embodiments, the polyethylene glycol is a monodisperse polyethylene glycol. In the context of the present specification, the term “monodisperse polyethylene glycol”, abbreviated also as “mdPEG”, refers to a polyethylene glycol (PEG) moiety that has a single, defined chain length and molecular weight. mdPEGs are typically generated by separation of individual chain length polymer species from the polymerization mixture by chromatography.
In certain embodiments, the compound is described by any one of formulae (7) to (13)
The solubility function, a poly(ethylene glycol) (PEG) chain shown here as a non-limiting example, is attached to the molecule to render it soluble in the colon lumen, at the concentrations of calcium present therein.
The compounds defined above can be synthesized according to standard methods. The synthesis of compounds (5a) and (6a) is described in the examples of the present invention.
According to another aspect of the invention, a compound according to any of the above aspects of the invention, in the broadest definition given, or as specified in any of the embodiments, is provided for use as a medicament. In particular examples of such embodiments, the medicament can contain pharmaceutically-acceptable carriers, salts, and/or excipients as known to the art.
According to yet another aspect of the invention, a compound according to any of the above aspects of the invention, in the broadest definition given, or as specified in any of the embodiments, is provided for use in methods for treatment or prevention of C. difficile infection. In such methods, the compound described herein (including a dosage form or formulation as described), is administered to a subject in need thereof, thereby treating or preventing the C. difficile infection. In particular embodiments, the methods include administration of additional antibiotic agents, in addition to the described compound, or as part of a combined treatment or dosage form, as described herein.
A compound according to the invention may be given to a patient already diagnosed with CDI, or to a patient being suspected of suffering from CDI. Alternatively, the compound may be used as a prophylactic for patients that are at risk of contracting the infection, such as patients under antibiotic treatment in hospital settings. The compounds according to the invention are simple to synthesize, resistant to degradation in the gastro-intestinal tract and unlikely to be absorbed into the bloodstream, thus avoiding potential side effects. The compounds according to the invention do not need to penetrate mammalian or bacterial membranes to be active, which makes them more effective in vivo. In addition, the compounds according to the invention are unlikely to exert selective pressure on the bacteria and therefore avoid problems related to resistance.
According to yet another aspect of the invention, a pharmaceutical composition for use in a method for the prevention or treatment of C. difficile infection is provided, comprising a compound according to any of the above aspects of the invention.
Particular pharmaceutical compositions comprise from approximately 1% to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.
A pharmaceutical composition according to the above aspects of the invention can be administered alone or in combination with one or more other therapeutic agents. A combination therapy may take the form of fixed combinations of the compound of the invention and one or more other antibiotic agents. Administration may be staggered; alternatively drugs may be given independently of one another, or as a fixed combination.
According to a particular embodiment, a pharmaceutical composition comprises a compound of the invention according to any of the above aspects of the invention, and additionally metronidazole, vancomycin and/or fidaxomicin.
According to yet another aspect of the invention, a dosage form is provided comprising a compound according to any of the above aspects of the invention. A peroral formulation, particularly a tablet, syrup, solution, capsule or powder is preferred.
According to a particular embodiment, such a dosage form additionally comprises an antibiotically active compound, such as (by way of non-limiting example) metronidazole, vancomycin or fidaxomicin.
According to yet another aspect of the invention a treatment regime is provided for the prevention and treatment of CDI, comprising the administration of a compound according to the invention. Administration may be effected by any of the means described herein.
Also within the scope of the present invention is a method for the prevention or treatment of CDI, comprising the administration a compound according to the invention to a subject in need thereof.
The compound according to the invention can also be used in the therapy or prevention of conditions related to pathological calcium crystallization.
Physiological calcium and phosphate concentrations in the blood are close to supersaturation. Blood components such as fetuin-A interact with calcium and phosphate to form soluble nanoparticles termed calciprotein particles (CPPs) that prevent precipitation and resultant calcification under normal conditions. So-called primary CPPs are amorphous and have a hydrodynamic radius of typically less than 100 nm and mature with time to reorganize into crystalline secondary CPPs that have a hydrodynamic radius of more than 100 nm. Secondary CPPs are subsequently thought to progress to calcification and to initiate pathological responses.
Pharmaceutical agents capable of reducing the propensity for progression of primary CPPs to secondary CPPs, and hence ultimately capable of reducing pathological crystallization are of significant therapeutic value. The inventors show that the compounds according to the invention are capable of reducing pathological calcium crystallization.
Conditions related to pathological calcium crystallization for which the compounds of the present invention are particularly useful include vascular calcification, coronary artery disease, vascular stiffening, valvular calcification, nephrocalcinosis, calcinosis cutis, kidney stones, chondrocalcinosis, osteoporosis, myocardial infarction, cardiovascular mortality (particularly in chronic kidney disease patients), progression of chronic kidney disease and failure of renal transplant grafts. Pathological crystallization has been shown to be associated with all-cause mortality of chronic kidney disease CKD) patients, hence the compounds of the present invention are indicated for chronic kidney disease patients in general.
One embodiment of the invention that shows a particularly high activity is the use of 2-PEG-IP5 (myo-pentakis-inositolphosphate-(2)-PEG) with PEG moieties having a molar mass of approximately 400 g/mol (n=9) or approximately 2000 g/mol (n=45) for the indications listed above. This conclusion is drawn from the results of an in vitro assay that measures the propensity for calcification of human serum, which has been clinically validated as a predictor of all-cause mortality in CKD patients and renal transplant patients (
Wherever alternatives for single features such as R1, R2, X etc. are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the entire molecule provided as such or for use in a method or medical indication herein. Thus, any of the alternative embodiments for R1 may be combined with any of the alternative embodiments of X provided in the formulae mentioned herein.
1. Synthesis of Compound (7)
The synthesis followed the sequence depicted in
Compound B: To a suspension of sodium hydride (4.3 mmol, 103.7 mg) in 10 mL dimethylformamide (DMF) was added a solution of compound A [Martin, S. F. et al., J. Org. Chem. 1994, 59, 4805] (2.16 mmol, 1363 mg) in DMF (10 mL) dropwise. When the addition was complete the mixture was stirred for 30 min at room temperature, followed by addition of MeO-PEG-OTs (OTs being toluenesulfonate) (3.2 mmol, 1.64 g in 10 mL DMF). The reaction was allowed to stir overnight, then quenched with water (5 mL). The mixture was extracted with dichloromethane (DCM). The solvent was evaporated and the residue was chromatographed on silica gel with six 100 mL portions of 20:80; 30:70; 40:60; 60:40; 80:20; 100:0 of ethylacetate:hexanes. The chromatography resulted in the fractionation of product with different PEG sizes, including compound B with an average of 9 PEG units. 1H NMR (400 MHz; CDCl3): δ 7.29-7.13 (m, 25H), 4.83 (d, J=10.8 Hz, 2H), 4.79 (s, 2H), 4.74 (d, J=10.8 Hz, 2H), 4.63 (d, J=11.7 Hz, 2H), 4.59 (d, J=11.6 Hz, 2H), 3.97-3.88 (m, 5H), 3.64-3.42 (m, 31H), 3.38 (t, J=9.2 Hz, 1H), 3.29-3.25 (m, 5H).
Compound C: Compound B was dissolved in a mixture of tetrahydrofuran (THF, 4 mL), methanol (7 mL) and water (3 mL), followed by addition of excess 10% palladium on charcoal. The mixture was placed under a hydrogen atmosphere and stirred overnight at room temperature. The reaction mixture was then purged with nitrogen, filtered and the solvent evaporated. The crude mixture was purified on a reverse phase cartridge (Sep-Pak, Waters, 1 g, C18, Cat.# WAT 036905) by eluting with 10 mL water. All fractions (1.5 ml) were lyophilized and analyzed by 1H NMR analysis. 1H NMR (400 MHz; D2O): δ 3.96-3.94 (m, 2H), 3.89 (t, J=2.8 Hz, 1H), 3.78-3.69 (m, 28H), 3.69-3.60 (m, 5H), 3.56 (dd, J=10.0, 2.8 Hz, 2H), 3.40 (s, 3H), 3.25 (t, J=9.2 Hz, 1H).
Compound D: Compound C (0.2 mmol, 119 mg) was suspended in tetrazole (3.630 mmol, 8.1 mL, 0.45 M in CH3CN) and DCM (10 mL) then N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (1.8 mmol, 434 mg) was added and the mixture was stirred at room temperature overnight. The mixture was then cooled to −10° C. and a solution of meta-chloroperoxybenzoic acid (mCPBA, predried over Na2SO4, 4.8 mmol, 1189 mg) in DCM (2 mL) was added. The mixture was allowed to stir at −10° C. for an additional 10 min, and then it was brought to room temperature and stirred for 1 hour. The mixture was washed with dilute sodium sulfite and extracted with DCM. The organic layers were dried with Na2SO4, filtered and concentrated. The residue was chromatographed on silica gel with a gradient of 1-5% methanol in DCM. 1H NMR (400 MHz; CDCl3): δ 7.41-7.27 (m, 18H), 7.21 (dd, J=6.9, 1.7 Hz, 2H), 5.59-5.51 (m, 6H), 5.44 (t, J=14.2 Hz, 2H), 5.37-5.29 (m, 6H), 5.25-4.95 (m, 10H), 4.74 (ddd, J=9.9, 7.6, 2.1 Hz, 2H), 4.61 (t, J=2.2 Hz, 1H), 4.03 (t, J=4.9 Hz, 2H), 3.68-3.50 (m, 22H), 3.48 (dd, J=6.1, 4.0 Hz, 2H), 3.40-3.34 (m, 7H). 13C NMR (101 MHz; CDCl3): δ 135.78, 135.72, 135.4, 135.14, 134.99, 129.38, 129.27, 129.26, 129.22, 129.14, 129.11, 128.96, 128.95, 77.6, 76.05, 76.01, 73.8, 71.9, 70.67, 70.60, 70.57, 70.54, 70.53, 70.49, 70.38, 70.34, 70.29, 69.46, 69.39, 69.33, 69.24, 69.16, 69.01, 68.95, 59.0
Compound (7): Compound D was dissolved in a mixture of THF (1 mL), methanol (1.5 mL) and water (2 mL), followed by addition of excess 10% palladium on charcoal. the mixture was placed under an hydrogen atmosphere and stirred overnight at room temperature. The mixture was then purged with nitrogen, filtered and concentrated. The compound was brought at pH 7 by addition of dilute aqueous NaOH (170 μl, 0.1M). The residue was purified on a sephadex column (PD-10, GE Healthcare, Sephadex G-25 M, cat.#17-0851-01) by eluting with 10 ml of water. All fractions (1.5 mL) were lyophilized and analyzed by 1H NMR. The fractions containing product were purified further on a reverse phase cartridge (Sep-Pak, Waters, 1 g, C18, cat.# WAT 036905) by eluting with 10 mL water. All fractions (1.5 ml) were lyophilized and analyzed by 1H NMR analysis. 1H NMR (400 MHz; D2O): δ 4.44 (q, J=9.4 Hz, 2H), 4.20 (s, 1H), 4.16-4.10 (m, 3H), 4.06 (t, J=4.7 Hz, 2H), 3.83-3.63 (m, 26H), 3.40 (s, 3H). 31P NMR (162 MHz; D2O): δ 1.5, 1.2, 0.8
2. Determination of EC50 in Presence of 10 mM Ca2+
The compound to be tested was added to a recombinant His-tagged cysteine protease domain of C. difficile toxin B in the presence of 10 mM Ca2+ in 100 mM Tris pH7.4 and incubated for 2 h at 37° C. Cleaved protein fragments were separated by SDS-PAGE and visualized by Coomassie staining. The extent of cleavage quantified from protein band intensities using the ImageJ software package. Signals were normalized to cleavage of positive and negative controls.
The results of this assay for IP6 and compound (7) are shown in
3. Comparison of Cleavage Kinetics
The compound to be tested was added to the His-tagged cysteine protease domain of C. difficile toxin B (same sequence as given above) in presence of 10 mM Ca2+ in 100 mM Tris pH 7.4 and incubated for 24 h at 37° C., with aliquots removed at regular intervals. Cleaved protein fragments were separated, visualized and analyzed as indicated above. The results of this assay for IP6 and compound (7) are shown in
4. Synthesis of Compound (5a)
The synthesis followed the sequence depicted in
5. Synthesis of Compound (6a)
The synthesis followed the sequence depicted in
6. Calcification Assay
The inventors performed an in vitro assay that measures the propensity for calcification of human serum and has been clinically validated as a predictor of all-cause mortality in CKD patients and renal transplant recipients (as described in Pasch, Journal of the American Society of Nephrology 23, 1744-1752, 2012). The experiment was carried out by mixing a calcium solution, human pooled serum, the test compound at the final concentration indicated and a phosphate solution, and the transition time of primary to secondary calciprotein particles (CPPs) was measured at 37° C. using a nephelometer for up to 600 minutes (
| Number | Date | Country | Kind |
|---|---|---|---|
| 11007933 | Sep 2011 | EP | regional |
| 11007935 | Sep 2011 | EP | regional |
This is a Continuation-in-Part of co-pending U.S. patent application Ser. No. 14/347,649, filed Mar. 27, 2014, which is the U.S. National Stage of International Application No. PCT/EP2012/004088, filed Sep. 28, 2012, which in turn claimed the benefit of European Patent Application No. 11007935.7, filed Sep. 29, 2011 and European Patent Application No. 11007933.2, filed Sep. 29, 2011. The foregoing applications are incorporated by reference in their entirety herein.
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| 5278332 | Siren | Jan 1994 | A |
| 20100048730 | Li et al. | Feb 2010 | A1 |
| 20120258936 | Savidge | Oct 2012 | A1 |
| Number | Date | Country |
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| 54044901 | Apr 1979 | JP |
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| WO 2010049921 | May 2010 | WO |
| WO 2012138963 | Oct 2012 | WO |
| Entry |
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| English machine translation of JP 54044901A obtained Jun. 9, 2017. |
| Partial Translation of JP 54044901A, Irina Knizhnik, USPTO Alexandria, VA (Jan. 2017). |
| STN CAplus entry for JP 54044901A (1979). |
| G. Guttenberg et al., “Inositol Hexakisphosphate-dependent Processing of Clostridium Sordellii Lethal Toxin and Clostridium novyi—Toxin”, Journal of Biological Chemistry, vol. 286, No. 17, Apr. 29, 2011, p. 14779-4786. |
| Florin I et. al. “Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B”, Biochimica ET Biophysica Acta, Molecular Cell Research, Elsevier Science Publshers, Amsterday, NL, vol. 805, No. 2, Oct. 12, 1984, pp. 131-136. |
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| Number | Date | Country | |
|---|---|---|---|
| 20160280727 A1 | Sep 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14347649 | US | |
| Child | 15172171 | US |
