MEDICAL USES OF THIOL-FUNCTIONALIZED POLYGLYCEROL DERIVATIVES

Abstract

It is provided a method of treatment of a human or animal patient in need thereof to achieve a reduction of the viscosity of mucus of the patient which is carried out by administering a polyglycerol derivative having a linear or dendritic polyglycerol backbone and carrying at least one thiol group covalently bound to the polyglycerol backbone. It is further provided a method of treatment of a human or animal patient suffering from chronic sinusitis, asthma, chronic bronchitis, cystic fibrosis, chronic obstructive pulmonary disease, emphysema, bronchiectasis, chronic inflammatory bowel diseases, constipation, gastrointestinal malabsorption syndrome, irritable bowel syndrome, steatorrhea or diarrhea by administering a polyglycerol derivative. Further specific thiol-functionalized polyglycerol derivatives and a corresponding manufacturing method are provided.

Claims

1. A method of treatment of a human or animal patient in need thereof to achieve a reduction of the viscosity of mucus of the patient, the method comprising the following step: administering a polyglycerol derivative to the patient, the polyglycerol derivative-having a linear or dendritic polyglycerol backbone and carrying at least one thiol group covalently bound to the polyglycerol backbone.

2.-14. (canceled)

15. The method of claim 1, wherein the polyglycerol backbone carries a plurality of sulfate groups, wherein a degree of sulfation of the polyglycerol backbone is between 10 and 100%.

16. The method of claim 1, wherein the polyglycerol backbone carries 1 to 100 thiol groups per polyglycerol derivative molecule.

17. The method of claim 1, wherein the polyglycerol backbone is biodegradable.

18. The method of claim 1, wherein the at least one thiol group is bound to the polyglycerol backbone via a linker.

19. The method of claim 1, wherein the polyglycerol derivative corresponds to the following general formula (I): ##STR00034## wherein dPG denotes a dendritic polyglycerol backbone, lPG denotes a linear polyglycerol backbone, X is a residue chosen from the group consisting of ##STR00035## A is a single-charge anionic counter ion, R.sup.1, R.sup.2 are independently from each other —OH or —OSO.sub.3.sup.−K.sup.+, R.sup.3 is H or a peptide residue having 1 to 20 amino acid residues, R.sup.4 is a C.sub.1-C.sub.10 hydrocarbon chain being optionally interrupted by N, S, and/or O and being at least substituted in such a way to carry at least one thiol group, R.sup.5 is a peptidyl comprising 1 to 20 amino acid residues, wherein at least one amino acid residue is a cysteine residue, K is a single-charge cationic counter ion, m is an integer between 1 and 20, n is an integer between 5 and 5000, o is an integer between 1 and 16, and p is an integer between 0 and 5000.

20. A method of treatment of a human or animal patient suffering from chronic sinusitis, asthma, chronic bronchitis, cystic fibrosis, chronic obstructive pulmonary disease, emphysema, bronchiectasis, chronic inflammatory bowel diseases, constipation, gastrointestinal malabsorption syndrome, irritable bowel syndrome, steatorrhea or diarrhea and being in need of such treatment by administering a polyglycerol derivative to the patient, the polyglycerol derivative having a linear or dendritic polyglycerol backbone and carrying at least one thiol group covalently bound to the polyglycerol backbone.

21. The method of claim 20, wherein the polyglycerol backbone carries a plurality of sulfate groups, wherein a degree of sulfation of the polyglycerol backbone is between 10 and 100%.

22. The method of claim 20, wherein the polyglycerol backbone carries 1 to 100 thiol groups per polyglycerol derivative molecule.

23. The method of claim 20, wherein the polyglycerol backbone is biodegradable.

24. The method of claim 20, wherein the at least one thiol group is bound to the polyglycerol backbone via a linker.

25. The method of claim 20, wherein the polyglycerol derivative corresponds to the following general formula (I): ##STR00036## wherein dPG denotes a dendritic polyglycerol backbone, lPG denotes a linear polyglycerol backbone, X is a residue chosen from the group consisting of: ##STR00037## A is a single-charge anionic counter ion, R.sup.1, R.sup.2 are independently from each other —OH or —OSO.sub.3.sup.−K.sup.+, R.sup.3 is H or a peptide residue having 1 to 20 amino acid residues, R.sup.4 is a C.sub.1-C.sub.10 hydrocarbon chain being optionally interrupted by N, S, and/or O and being at least substituted in such a way to carry at least one thiol group, R.sup.5 is a peptidyl comprising 1 to 20 amino acid residues, wherein at least one amino acid residue is a cysteine residue, K is a single-charge cationic counter ion, m is an integer between 1 and 20, n is an integer between 5 and 5000, o is an integer between 1 and 16, and p is an integer between 0 and 5000.

26. A polyglycerol derivative corresponding to the following general formula (I): ##STR00038## wherein dPG denotes a dendritic polyglycerol backbone, lPG denotes a linear polyglycerol backbone, X is a residue chosen from the group consisting of: ##STR00039## R.sup.1, R.sup.2 are independently from each other —OH or —OSO.sub.3.sup.−K.sup.+, R.sup.3 is H or a peptide residue having 1 to 20 amino acid residues, R.sup.4 is a C.sub.1-C.sub.10 hydrocarbon chain being optionally interrupted by N, S, and/or O and being at least substituted in such a way to carry at least one thiol group, R.sup.5 is a peptidyl comprising 1 to 20 amino acid residues, wherein at least one amino acid residue is a cysteine residue, K is a single-charge cationic counter ion, m is an integer between 1 and 20, n is an integer between 5 and 5000, is an integer between 1 and 16, and p is an integer between 0 and 5000.

27. The polyglycerol derivative according to claim 26, wherein the polyglycerol derivative comprises a dendritic polyglycerol backbone.

28. The polyglycerol derivative according to claim 26, wherein X is chosen from the group consisting of: ##STR00040##

29. The polyglycerol derivative according to claim 26, wherein o is 1.

30. A method for manufacturing a polyglycerol derivative according to claim 29, comprising the following steps: a) adding a solution of mercaptoacetic acid and an activation reagent in water or a polar aprotic solvent to a solution of an amine-functionalized polyglycerol derivative corresponding to the following general formula (II) in water or a polar aprotic solvent: ##STR00041## wherein dPG denotes a dendritic polyglycerol backbone, lPG denotes a linear polyglycerol backbone, Y is —NH.sub.2, R.sup.1, R.sup.2 are independently from each other —OH or —OSO.sub.3.sup.−K.sup.+, K is a single-charge cationic counter ion, m is an integer between 1 and 20, n is an integer between 5 and 5000, and p is an integer between 0 and 5000, b) letting react all compounds and removing the solvent to obtain a thiol-functionalized polyglycerol derivative corresponding to the following general formula (I): ##STR00042## wherein X is ##STR00043## wherein o is 1.

31. The method according to claim 30, wherein the amine-functionalized polyglycerol derivative and the thiol-functionalized polyglycerol derivative are dendritic polyglycerol derivatives.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Further details of aspects of the solution will be explained with respect to exemplary embodiments and accompanying Figures.

[0126] FIG. 1 shows synthetic routes for manufacturing different thiol-containing polymers.

[0127] FIG. 2A shows a .sup.1H NMR spectrum (600 MHz, D.sub.2O) of dPGS-NH.sub.2.

[0128] FIG. 2B shows a .sup.1H NMR spectrum (600 MHz, D.sub.2O) of dPGS-SH.

[0129] FIG. 3 shows a calibration curve of 3-mercaptopropionic acid solution.

[0130] FIG. 4A shows a .sup.1H NMR spectrum (600 MHz, D.sub.2O) of dPG-NH.sub.2.

[0131] FIG. 4B shows a .sup.1H NMR spectrum (600 MHz, D.sub.2O) of dPG-SH.

[0132] FIG. 5 shows a .sup.1H NMR spectrum (600 MHz, D.sub.2O) of dPGS-LA(SH).

[0133] FIG. 6 shows a synthetic route for manufacturing dPGS-mercaptoacetic acid.

[0134] FIG. 7 shows the influence of a treatment of cystic fibrosis mucus with different mucolytics on the elastic modulus of the mucus.

[0135] FIG. 8 shows the influence of a treatment of cystic fibrosis mucus with different mucolytics on the viscosity of the mucus.

[0136] FIG. 9 shows a reaction scheme of the synthesis of lPG-dithiol via mesylation.

[0137] FIG. 10 A shows half-wave potential of DTT and thiol-containing polyglycerols from cyclic voltammetry measurements.

[0138] FIG. 10 B shows the thiol concentration of two batches of dPGS-SH after synthesis, and after a 10-month storage as lyophilisate at 4° C.

[0139] FIG. 10 C shows a Western blot of mucus samples from bronchoalveolar lavage (BAL) of βENaC-Tg mice subjected to increasing concentrations of dithiothreitol (DTT), N-acetylcysteine (NAC) and dPGS-SH (0.2-20 mM).

[0140] FIG. 10 D shows the elastic moduli determined by macrorheology on unprocessed mucus from healthy persons or CF patients from a shear strain sweep (φ=1 Hz).

DETAILED DESCRIPTION

[0141] Synthesis of dPGS-SH

[0142] Dendritic polyglycerol sulfate-thiol (dPGS-SH) was obtained via the reaction between dPGS-amine (dPGS-NH.sub.2, 38.5 kDa, sulfate: 81.7 mol. %, amine: 10.7 mol. %) and 2-iminothiolane hydrochloride (Sigma, 98%) (FIG. 1, reaction scheme A). In general, to a stirred solution of dPGS-NH.sub.2 (1.0 g, 20 mL) in degassed Milli Q water, a solution of 2-iminothiolane hydrochloride (112 mg in 3 mL of the same solvent system, 1.2 eq.) was added. The reaction continued at room temperature (r.t.) for 1 h with a pH of 7.0 by adding KOH solution. Afterwards, the target polymer dPGS-SH was purified via ultrafiltration, and obtained as light yellow powder after lyophilization. The yield was 87%.

[0143] The characterization of dPGS-SH was evaluated by .sup.1H NMR and Ellman's test. From FIG. 2 (compare the differences between FIG. 2A and FIG. 2B), besides the peaks of dPGS backbone, the appearance of δ 2.04, 2.41, and 2.61 indicated the successful formation of dPGS-SH. .sup.1H NMR (600 MHz, D.sub.2O): δ 3.19-4.34, 4.56-4.74 (dPGS); δ 2.04, 2.41, and 2.61 (2-iminothiolane).

[0144] To quantitate the thiol content of the polymers, an Ellman's test was performed. According to the calibration curve from a series of 3-mercaptopropionic acid solution in PBS (ranging from 0.0039 to 0.125 mM), an equation Y=9.6322X+0.3554 was obtained (FIG. 3), giving a thiol content of about 1.57 mol. % for dPGS-SH.

[0145] Synthesis of dPG-SH

[0146] Dendritic polyglycerol-thiol (dPG-SH) was obtained via the reaction between dPG-amine (dPG-NH.sub.2, 10.0 kDa, amine: 10.0 mol %) and 2-iminothiolane hydrochloride (Sigma, 98%) (FIG. 1, reaction scheme B). In general, to a stirred solution of dPG-NH.sub.2 (0.5 g, 8 mL) in degassed Milli Q water, a solution of 2-iminothiolane hydrochloride (42 mg in 2 mL of the same solvent system, 1.2 eq.) was added. The reaction continued at r.t. for 1 h with a pH of 7.0 by adding KOH solution. Afterwards, the target polymer dPG-SH was purified via ultrafiltration, and obtained as light yellow oil after lyophilization. The yield was 85%.

[0147] The characterization of dPG-SH was evaluated by .sup.1H NMR and Ellman's test. From FIG. 4 (compare the differences between FIG. 4A and FIG. 4B), besides the peaks of dPG backbone, the appearance of δ 2.19 indicated the successful formation of dPG-SH. .sup.1H NMR (600 MHz, D.sub.2O): δ 2.69, 2.77, 3.05-4.01 (dPG); δ 2.19 (methylene proton next to imine).

[0148] To quantitate the thiol content of the polymers, we also performed Ellman's test. According to the calibration curve from a series of 3-mercaptopropionic acid solution in PBS (ranging from 0.0039 to 0.125 mM), the equation Y=10.411X+0.3504 was obtained, giving a thiol content of about 0.53 mol. % for dPG-SH.

[0149] Synthesis of dPGS-LA(SH)

[0150] The dithiol-containing dPGS was obtained in the following three steps (FIG. 1, reaction scheme IC): i) the formation of lipoic acid anhydride (LAA) at the presence of dicyclohexylcarbodiimid (DCC), ii) the reaction between LAA and dPGS-NH.sub.2 via the catalysis of N,N-4-dimethylaminopyridine (DMAP), and iii) the breakage of the disulfide bond in LA at the presence of dithiothreitol (DTT). Firstly, under an argon atmosphere, to a stirred aqueous solution of lipoic acid (219 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl), a solution of DCC (138 mg, 1.1 eq.) in 6 mL water was dropwisely added. The reaction was allowed to proceed under stirring for 12 h at r.t. in the dark. The precipitate generated during the reaction was removed by filtration, and the filtrate was concentrated via rotary evaporation to give LAA. The obtained LAA was then dissolved in 5 mL DMSO, and added into the solution of dPGS-NH.sub.2 (0.6216 g) in 10 mL DMSO at the presence of DMAP (120 mg). The reaction proceeded under stirring for 48 h at room temperature in the dark. Finally, DTT (246 mg) was added into the resulting solution and reacted for 24 h under stirring at r.t. The target polymer dPGS-LA(SH) was purified via dialysis in degassed deionized water under an argon atmosphere for 48 h, and freeze-dried. The yield was 74%. .sup.1H NMR (600 MHz, D.sub.2O): δ 1.35, 1.58, 1.94, 2.23-2.43, 2.67 (LA); δ 3.19-4.26, 4.44-4.64 (dPGS) (FIG. 5).

[0151] Synthesis of dPGS-LA(SH) with Lower Conjugation

[0152] Firstly, under an argon atmosphere, to a stirred solution of lipoic acid (110 mg) and EDCl in 3 mL water, a solution of DCC (69 mg, 1.1 eq.) in 3 mL water was dropwisely added. The reaction was allowed to proceed under stirring for 12 h at r.t. in the dark. The precipitate generated during the reaction was removed by filtration, and the filtrate was concentrated via rotary evaporation to give LAA. The obtained LAA was then dissolved in 5 mL DMSO, and added into the solution of dPGS-NH.sub.2 (0.6216 g) in 10 mL DMSO at the presence of DMAP (60 mg). The reaction proceeded under stirring for 48 h at room temperature in the dark. Finally, DTT (123 mg) was added into the resulting solution and reacted for 24 h under stirring at r.t. The target polymer dPGS-LA(SH) was purified via dialysis in degassed deionized water under an argon atmosphere for 48 h, and freeze-dried.

[0153] It is apparent that all reactions according to the reaction schemes A, B, and C of FIG. 1 could likewise be performed in the same manner with a linear polyglycerol or a linear polyglycerol sulfate.

[0154] Stability of dPGS-SH

[0155] The stability of the manufactured dPGS-SH was tested by determining its thiol content immediately after manufacturing the compound and once again after a plurality of months (6 months or 10 months, respectively). According to the results depicted in Table 1, no significant difference between the individual measurements of the same sample could be observed. Thus, the thiol group remains stable in dPGS-SH even after a prolonged storage.

TABLE-US-00002 TABLE 1 Stability of dPGS-SH on the basis of thiol content at different time points. Thiol Content (mol. %) Theoretic Determined Samples maximum Determined (Apr. 17, 2019) dPGS-SH (Jun. 4, 2018) 10 1.31 1.30 dPGS-SH (Oct. 12, 2018) 10 1.57 1.57

[0156] Further Thiol-Containing dPG/IPG/dPGS/IPGS

[0157] There are a series of other thiol-functionalized alternatives of the previously described dPGS-SH like dPG(S)-mercapto carboxylic acid, dPG(S)-DTT, dPG(S)-β-mercaptoethanol, dPG(S)-cysteamine and dPG(S)-cysteine to name only a few.

[0158] For synthesis of dPGS-mercaptoacetic acid (a specific example of dPG(S)-mercapto carboxylic acid), the conjugation is conducted by adding a solution of mercaptoacetic acid (35 mg, 4.5 eq.) and EDCl (94.7 mg, 5.9 eq.) in DMSO (15 mL) to a solution of dPGS-amine (1 g, 0.084 mmol) in DMSO (5 mL). The mixture is then stirred at 50° C. for 4 h and at r.t. for 24 h. In the end, the solvent will be evaporated under reduced pressure, and the raw product is dissolved in degassed Milli Q water, dialyzed against the same media for three days, and lyophilized to remove water.

[0159] The reaction scheme is depicted in FIG. 6. It is apparent that the synthesis of dPG-mercaptoacetic acid can be achieved in the same manner as the synthesis of dPGS-mercaptoacetic acid. The reaction could likewise be performed in the same manner with a linear polyglycerol or a linear polyglycerol sulfate.

[0160] Biomechanical Studies on Mucus Using Macrorheology

[0161] In order to determine the mucolytic potential of thiol-functionalized polyglycerol derivatives the rheological properties of lung mucus, which was collected from patients with cystic fibrosis, were determined before and after treatment with different reducing or control agents. In addition, mucus from a healthy donor was used as control. Rheological measurements can be generally used to determine both elastic and viscous material components.

[0162] Clinical samples were obtained freshly from cystic fibrosis (CF) patients at Charité Berlin and put directly on ice after collection. Then, the samples were mixed with a protease inhibitor cocktail (Roche) and aliquoted in 150 μl portions. The samples were then treated with different concentrations of freshly prepared reducing agents for 30 min at 37° C. Control samples were kept on ice throughout the incubation time or treated with phosphate-buffered saline (PBS) and incubated for 30 min at 37° C. After the incubation time all samples were kept on ice until use for the macrorheological studies. Here, a Kinexus instrument (Malvern) with a cone-and-plate setup was used. The cone geometry was 20 mm diameter and a 1° C. angle. Each sample was run with an amplitude sweep mode at an oscillatory frequency of 1 Hz to determine the linear viscoelastic regime. In order to obtain viscosity parameters, measurements were repeated at 2% stain in a frequency sweep mode.

[0163] The results are shown in FIG. 7. Dominant elastic components were found for both biomaterials from the elastic modulus for (G′). A treatment of CF mucus with N-acetylcysteine (NAC) showed a comparably slight reduction of the elastic modulus like a treatment with physiological phosphate buffer (PBS).

[0164] A treatment of CF mucus with thiol-functionalized dendritic polyglycerol sulfate (here referred to as dPGS, corresponding to dPGS-SH according to reaction scheme A of FIG. 1 and the corresponding explanations above) reduced the elastic modulus of CF mucus almost to the level of untreated, healthy mucus.

[0165] Dithiothreitol (DTT) was used as a positive control. It is also referred to as sputolysin and is used in order to further prepare mucus samples for analytical purposes. Due to its high toxicity, however, it is not suitable for clinical use. Even if it was not toxic, it appears to reduce almost all disulfide bonds of the mucus and thus reduces the viscosity of CF mucus to level significantly below the viscosity level of mucus from healthy individuals. Thus, DTT destroys the natural functionality of mucus which is, in contrast, preserved after a treatment with dPGS-SH.

[0166] FIG. 8 shows a depiction of the viscosity over the frequency and confirms the results explained with respect to FIG. 7. Briefly, treatment of CF mucus with dPGS-SH results in a mucus viscosity that corresponds to the mucus viscosity of healthy mucus. In contrast, NAC does not show any enhanced mucolytic activity than PBS. Furthermore, DTT completely destroys the viscosity properties of mucus and thus impairs the physiologic functionality of mucus.

[0167] Synthesis of lPG-Dithiol Via Mesylation

[0168] The four-step reaction scheme of this synthesis is depicted in FIG. 9. It will be briefly explained in the following. Notably, this synthesis is not limited to linear polyglycerol backbones but generally works with almost every hydroxyl functionalization, i.e. in particular also with dendritic polyglycerol backbones.

[0169] Step I: Cleavage of Bromine

[0170] The —Br residue on the backbone of compound (a) was cleaved by stirring the compound overnight in sodium methoxide (1M solution from 4.4 M solution, 200 μL)+Methanol 18 mL+H.sub.2O 3 mL.

[0171] The resulting lPEEGE (b) was purified by a 1.5-days dialysis against acetone.

[0172] Step II: Mesylation

[0173] Compound (b) was thoroughly dried for the mesylation step and dissolved in dry dimethylformamide (DMF). 7.5 equivalents of trimethylamine were added to the solution under stirring. Afterwards, ensuring the temperature was maintained at 0° C. using an ice bath, 3 equivalents of mesyl chloride solution (MsCl) in dry DMF were added dropwise. The ice bath was removed and the reaction was allowed to carry on overnight.

[0174] After ensuring the pH was ˜8-10 (triethanolamine (TEA) was optionally used for pH adjustment), the resulting compound (c) was then dialyzed against methanol for 2 days (3 changes per day). NMR analysis confirmed formation of compound (c).

[0175] Step III: Thiolation by Thiourea

[0176] To a solution of (c) in methanol and 1-propanol, 4 equivalents of thiourea were added. With a condenser (and some needles as outlet), the reaction mixture was heated up to 115° C., and continued overnight (˜1.5 d). Analysis of the resulting compound (d) was carried out by CHNS analysis.

[0177] Step IV: Deprotection

[0178] Using 4 equivalents of sodium hydroxide, with water as solvent, compound (d) was refluxed under the same conditions overnight.

[0179] The pH of the resulting solution was brought to 5-6. To obtain the purified compound (e), dialysis was carried out against water for 2 days (3 changes).

[0180] Further Results Regarding the Effects of dPGS-SH on Mucus

[0181] FIGS. 10 A to 10 D show the results of studies on the effects of dPGS-SH on biochemical and rheological properties of mucus from mice with CF-like lung disease and patients with CF.

[0182] After synthesis of the polymer-based reducing agents (dPG-SH and dPGS-SH), redoxpotentials were determined using cyclic voltammetry and compared to that of DTT (FIG. 10 A). For this purpose, the compounds were measured at 5 mM thiol content in DMSO against a ferrocinium/ferrocene reference electrode. At equimolar thiol concentrations the dPG(S)-SH systems demonstrated a higher redoxpotential than DTT.

[0183] In addition, the thiol concentration was determined with the Ellman's assay (using 5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB as reagent) immediately after synthesis and 10 months later (FIG. 10 B). Upon storage as lyophilisate at 4° C. the thiol-content of dPGS-SH did not change significantly for a period of 10 months. Regarding the compound stability, this aspect is an important finding.

[0184] Following, the different reducing agents were tested in a mucolytical activity assay on mucus from mice with CF phenotype using a Western blot analysis (FIG. 10 C). The samples were incubated at 37° C. for 30 minutes and then quenched. Western blotting was performed to assess the mucolytic potency of the three different reducing agents. The results indicate that dPGS-SH was more active than the clinically approved NAC at 2 mM and at 20 mM. Beyond, by comparing the 20 mM lanes of DTT and NAC, different molecular weight distributions of proteins could be observed. This indicates the stability of specific disulfide bonds towards certain reducing agents in the mucin network.

[0185] Finally, the mucolytic activity was probed on mucus samples from CF patients (FIG. 10 D). The collected mucus was kept on ice or treated for 30 minutes at 37° C. with the indicated reducing agent or a buffer control. After the treatment, the samples were quenched with 50 mM iodoacetamide and put on ice until they underwent rheological measurements. Here, the storage modulus of human mucus could be decreased with all reducing agents. The reducing efficacy of dPGS-SH was lower than that of DTT, but higher than the clinically approved drug NAC at equimolar concentrations.