Therapy for transthyretin-associated amyloidosis

Abstract

It is provided a catechol-O-methyltransferase (COMT) inhibitor for use in the prevention and/or treatment of transthyretin-associated amyloidosis. It is also provided a catechol-O-methyltransferase (COMT) inhibitor for use in the prevention and/or treatment of transthyretin-associated amyloidosis in combination therapy with another COMT inhibitor, a benzoxazole derivative, iododiflunisal, diflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline, or epigallocatechin-3-gallate.

Claims

1. A method for treating transthyretin (TTR)-associated amyloidosis comprising administering to a subject in need thereof a dose of between 0.1 mg and 16,000 mg per day of at least one catechol-O-methyletransferase (COMT) inhibitor of Formula I or a pharmaceutically acceptable salt thereof ##STR00007## wherein R is selected from the group consisting of —C(O)—PhCH.sub.3, —CH=C(CN)—C(O)—NEt.sub.2 and —CH=C(C(O)CH.sub.3).sub.2.

2. The method of claim 1, wherein the dose is between 0.1 mg and 16,000 mg per day.

3. The method of claim 1, wherein the dose is between 0.1 mg and 12,000 mg per day.

4. The method of claim 1, wherein the dose is between 0.1 mg and 10,000 mg per day.

5. The method of claim 1, wherein the dose is between 0.1 mg and 5,000 mg per day.

6. The method of claim 1, wherein the dose is between 1 mg and 3,000 mg per day.

7. The method of claim 1, wherein the dose is between 1 mg and 2,000 mg per day.

8. The method of claim 1, wherein the COMT inhibitor of Formula I is tolcapone or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the dose is between 20 mg and 600 mg per day.

10. The method of claim 1, wherein the transthyretin-associated amyloidosis is familial amyloid polyneuropathy.

11. The method of claim 1, wherein the transthyretin-associated amyloidosis is familial amyloid cardiomyopathy.

12. The method of claim 1, wherein the transthyretin-associated amyloidosis is senile systemic amyloidosis.

13. The method of claim 1, wherein the transthyretin-associated amyloidosis is leptomeningeal amyloidosis.

14. The method of claim 1, wherein the at least one COMT inhibitor is administered as an injectable dosage form, as an oral dosage form or as a controlled release dosage form.

15. The method of claim 14, wherein the oral dosage form is selected from the group consisting of a tablet, pill, powder, capsule, sachet, liquid syrup, suspension and elixir.

16. The method of claim 1, further comprising administering an additional therapeutic agent selected from the group consisting of another COMT inhibitor as defined in claim 14, a benzoxazole derivative, iododiflunisal, diflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline and epigallocatechin-3-gallate, wherein the benzoxazole derivative is a compound of Formula V ##STR00008## or a pharmaceutically acceptable salt thereof, wherein: Y is selected from the group consisting of COOR, tetrazolyl, CONHOR, B(OH).sub.2 or OR; X is O; and R.sub.1, R.sub.2 and R.sub.3 are each independently selected from the group consisting of hydrogen, halo, OR, B(OH).sub.2 or CF.sub.3, and R is hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 cycloalkyl, C1-C6 heterocyclyl, phenyl, xylyl, naphthyl, thienyl, indolyl or pyridyl.

17. The method of claim 16, wherein the benzoxazole derivative is a compound of formula VI ##STR00009## or a pharmaceutically acceptable salt thereof, wherein: Y is selected from the group consisting of COOH, and OH; and R.sub.1, R.sub.2 and R.sub.3 are each independently selected from the group consisting of hydrogen, halo, OH, B(OH).sub.2 and CF.sub.3.

18. The method of claim 16, wherein the benzoxazole derivative is tafamidis.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Assay of competition with T4 for the binding to TTR wild type (WT) by gel filtration: Curves of T4 displacement from TTR WT by different compounds. Y axis: Amount of TTR-bound T4/total T4; X-axis: log 10 concentration of compound (molar units). Values correspond to a representative experiment done in duplicates, represented as average+/−standard deviation. Test compounds: Thyroxine (T4), Tolcapone (SOM), Tafamidis (TAF), and (−)-epigallocatechin-3-gallate (EGCG).

(2) FIG. 2. TTR tetrameric stability in the presence of different compounds by IEF: Plasma from control individuals (C) and from familial amyloid polyneuropathy patients carrying V30M mutation (V30M) was treated with test compounds Tafamidis (T); tolcapone (S); epigallocatechin-3-gallate (EGCG) or left untreated (nt); and subjected to IEF under semidenaturing conditions as described in the text. The ratio of TTR tetramer/total TTR for each condition was calculated and represented as average+/−sem (standard error of the mean).

(3) FIG. 3: Caspase-3 activation. Rat Schwannoma cells (RN22 cell line) were incubated 24 h in the absence or presence of TTR Y78F oligomers obtained in the absence or presence of tested compounds (at 20 μM). Activation of Caspase-3 was measured in cell lysates, and expressed as fluorescence/protein content. Samples: control cells (C1); Cells treated with EGCG (C2); Cells treated with tafamidis (C3); cells treated with tolcapone (C4); control cells treated with oligomer obtained in the absence of compounds (O1); cells treated with oligomer obtained in the presence of EGCG (O2); cells treated with oligomer obtained in the presence of tafamidis (O3); cells treated with oligomer obtained in the presence of tolcapone (O4). Results represent average of 4 replicates and standard deviation. Significant differences respect O1 control were calculated with T-student test: *: P<0.05; ***: P<0.005.

(4) FIG. 4: Transmission Electron Microscopy analysis of preformed TTR fibrils after 4 days incubation with different compounds at 36 μM. From up left, clockwise: control, tafamidis, EGCG, Tolcapone.

EXAMPLES

Example 1: Kinetic Turbidity Assay

(5) Materials

(6) Recombinant Y78F TTR protein, which is a Tyr78Phe highly amyloidogenic variation of human TTR, was produced as reported in Dolado et al (Dolado I, Nieto J, Saraiva M J, Arsequell G, Valencia G, Planas A. “Kinetic Assay for High-Throughput Screening of In Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors”. J. Comb. Chem., 2005, vol. 7, p. 246-252).

(7) Tolcapone was obtained from Santa Cruz Biotechnology, Inc. lododiflunisal, was prepared from diflusinal (Sigma) by reaction with bis(pyridine)iodonium tetrafluoroborate (IPy.sub.2BF.sub.4) as described by Barluenga et al (Barluenga J, Gonzalez J M, Garcia-Martin M A, Campos P J, Asensio G. “An expeditious and general aromatic iodination procedure. J Chem Soc Chem Commun, 1992, vol. 14, p. 1016-1017). Tafamidis can be prepared by the methods disclosed in the international patent application WO2005113523. Stocks of compounds assayed as inhibitors were dissolved in DMSO (spectrophotometry grade from Sigma) at 1.5 mM concentration. Working solutions were prepared by diluting the stock solution 1:4 in H2O/DMSO (2:1). In all cases, DMSO concentration was adjusted to 5% (v/v) in the final reaction assay mixture.

(8) Methods

(9) The assay was performed according to the procedure described in Dolado et al (supra). The assay comprises two stages, one stage where the Y78F protein is incubated together with the inhibitor during 30 minutes, and a second stage where fibril formation is induced by a change in pH and absorbance is measured along 1.5 h. Briefly, the assay was performed as follows:

(10) First, the following solutions were prepared: Protein Y78F stock: 4 mg/mL in 20 mM phosphate, 100 mM KCl, pH 7.6. Incubation buffer: 10 mM phosphate, 100 mM KCl, 1 mM EDTA, pH 7.6. Dilution buffer: 400 mM sodium acetate, 100 mM KCl, 1 mM EDTA, pH 4.2.

(11) For each inhibitor the following protocol was followed: Exact protein concentration of the stock solution was determined by Abs.sub.280 and according to this value, the volume of Y78F stock to be added to have a final protein well concentration of 0.4 mg/mL was calculated and dispensed into 6 wells of a 96-well microplate. Different volumes of working inhibitor solution were added to give final concentrations ranging from 0 to 40 μM, and the final DMSO content of each well was adjusted to 5% by adding the corresponding volume of a H.sub.2O/DMSO (1:1) solution. Incubation buffer was then added up to a volume of 100 μL. The plate was incubated at 37° C. in a thermostated microplate reader with orbital shaking 15 s every minute for 30 min. A 100 μL portion of dilution buffer was dispensed to each well, and the mixture was incubated at 37° C. with shaking (15 s every min) in the microplate reader. Absorbance at 340 nm was monitored for 1.5 h at 1 min intervals. Data were collected and analyzed using Microsoft Excel software. All assays were done in duplicate.

(12) Result Analysis

(13) After following the general procedure indicated above, time-course curves were obtained, from which the initial rates of fibril formation (V.sub.0) were calculated as the slopes of the linear increase of absorbance. When plotting the initial rates vs inhibition concentration, an exponential decay was obtained with all inhibitors analyzed. Data were fitted to equation (1):
V.sub.0=A+B*e.sup.−C[I]  (1),
where V.sub.0 is the initial rate of fibril formation (in absorbance units per hour, Abs*h.sup.−1), and [I] is the concentration of the inhibitor (μM). Adjustable parameters are A (Abs*h.sup.−1), residual aggregation rate at high concentration of inhibitor; B (Abs*h.sup.−1), amplitude or maximum decrease of initial rate of fibril formation; and C (μM.sup.−1), the exponential constant. A+B is equal to the initial rate of fibril formation under the assay conditions in the absence of inhibitor.

(14) The following parameters were estimated to evaluate the potency of a compound as fibril formation inhibitor: IC.sub.50: concentration of inhibitor at which the initial rate of fibril formation is one-half that without inhibitor. RA(%)=100*B/(A+B): percent reduction of fibril formation rate at high inhibitor concentration relative to the rate at [I]=0. Results of evaluation of the inhibition properties of assayed compounds are summarized in Table 1.

(15) TABLE-US-00001 TABLE 1 IC.sub.50 and percentage of amyloidosis reduction (RA) values for TTR fibril formation inhibitors Compound IC.sub.50 (μM) RA (%) Tolcapone 4.8 85.8 Iododiflunisal 3.9 99.8 Tafamidis 16.9 99

(16) It can be observed by the above results that tolcapone is an effective inhibitor of TTR fibril formation, as it showed a low IC.sub.50 and a high RA. According to their IC.sub.50 values, tolcapone has a similar inhibition capacity as compared with iododiflunisal, which has been reported as one of the most potent TTR fibril formation inhibitors in vitro. Further, according to the IC.sub.50, tolcapone is more effective than tafamidis, since it shows an IC.sub.50 which is four times lower than tafamidis. These results demonstrate that tolcapone is a promising drug for TTR-related amyloidosis, such as FAP, familial amyloid cardiomyopathy senile systemic amyloidosis and leptomeningeal amyloidosis.

Example 2: End-Point Turbidity Assay with a Familiar Amyloid Cardiomyopathy Mutant Variant of TTR

(17) Materials

(18) Recombinant V122I TTR protein, which is an amyloidogenic variation of human TTR associated with Familial Amyloid Cardiomyopathy (FAC), was produced by following the same procedure described for the Y78F variant used in Example 1. Plasmid DNA expressing the V122I mutant was prepared by site-directed mutagenesis as reported for Y78F in Dolado et al (supra). but using the following primers: 5′-GGATTGGTGATGACAGCCGT-3′ and 5′-ACGGCTGTCATCACCAATCC-3′. Tolcapone and lododiflunisal were obtained as described in Example 1.

(19) Methods

(20) This assay is used for TTR variants with lower amyloidegenicity than the Y78F variant when the kinetic turbidity assay is not sensitive enough for accurate measurements. The procedure followed to test the inhibitors by this end-point assay at 72 h is reported in Dolado et al, (supra). V122I TTR was incubated with the inhibitor under the same conditions described above for the kinetic turbidity assay (Example 1), using V122I protein at a concentration of 0.4 mg/mL and three different concentrations of inhibitor: 3.6, 7.2 and 21.8 microM, corresponding to 0.5×[protein], 1×[protein], and 3×[protein]. After acid induction (addition of dilution buffer), samples were incubated without shaking for 72 h at 37° C. and then homogenized by mixing to resuspend any fibrils present. Turbidity was measured at 340 nm and normalized to amyloidogenesis in the absence of inhibitor.

(21) Result

(22) The inhibitory potency of the tested compounds was evaluated as the percentage of absorvance reduction of the inhibitor-containing samples when compared with the inhibitor-free control sample.

(23) TABLE-US-00002 TABLE 2 % Fibril Reduction values for V122I TTR fibril formation inhibitors Inhibitor concentration: 0.5x[protein] 1x[protein] 3x[protein] Tolcapone 79.3% 84.3% 100.0% Iododiflunisal 83.2% 85.0% 88.2%

(24) % Fibril reduction=100×(1-turbidity sample/turbidity blank), where turbidity sample is the turbidity measured in the presence of inhibitor, and turbidity blank is that in the absence of inhibitor.

(25) The above results show that tolcapone effectively inhibits fibril formation by V122I mutant ATTR, even at a inhibitor:protein molar ratio of 1:2 (0.5×[protein]). According to these values, tolcapone has a similar inhibition capacity as compared with iododiflunisal. These results demonstrate that tolcapone is a promising drug for TTR-related amyloidosis, including familial amyloid cardiomyopathy, which is caused mainly by the V122I mutation.

Examples 3-6

Materials for Examples 3-6

(26) Tolcapone and tafamidis were obtained as described in example 1. The Epigallocatechin-3-gallate (EGCG, CAS No. 989-51-5) was purchased from Cayman Chemicals (#70935). Recombinant wild-type TTR (TTR WT), TTR Y78F and TTR L55P variants were produced in a bacterial expression system using Escherichia coli BL21. Recombinant TTRs were isolated and purified as previously described (Ferreira et al, 2009, FEBS Lett, vol. 583, p. 3569-76). Whole blood from TTR V30M heterozygote carriers and from control individuals were obtained from a collection of samples available at the Molecular Neurobiology Group, IBMC (University of Porto). Blood samples had been collected in the presence of EDTA and centrifuged for the separation of plasma. Plasmas had been kept frozen at −20° C.

Example 3: Assay of Competition with Thyroxine (T4) for the Binding to TTR Wild Type (WT) by Gel Filtration

(27) Binding of small molecule ligands to the T4 binding sites of TTR might stabilize the TTR tetramer and slow tetramer dissociation and amyloidogenesis in vitro. To asses binding, competition of test compounds with T4 (Sigma-Aldrich) for binding to TTR WT was assayed quantitatively by a gel filtration procedure, using a constant amount of TTR (100 μL of 60 nM solution) incubated with a trace amount of radiolabeled [125I]T4 (corresponding to 50.000 cpm; 125I-T4 specific activity 1250 μCi/μg from Perkin-Elmer, MA, USA) and with 100 μL of solution of either test compounds or T4 (positive control) at different concentrations, namely 0, 20, 60, 200, 600, 2000 6000 and 20000 nM (0-10 μM final concentration) (Ferreira et al, 2011, FEBS Lett., vol. 585, p. 2424-30). The negative control was prepared with the protein, plus labelled T4 plus 100 μL of THE (absence of competitor). All solutions were prepared in THE buffer (Tris 0.1 M, NaCl 0.1 M, EDTA 1 mM). All samples were prepared in duplicate. Radioactivity was measured in each sample, in a gamma scintillation counter Wizard 14701, Wallac. The samples were then incubated overnight at 4° C. After incubation, T4 bound to TTR was separated from unbound T4 by filtration through a P6DG gel filtration column (1 mL, BioRad). Radioactivity was measured in the eluted samples. The results were expressed as the amount of TTR-bound T4/total T4 against Log total concentration of test compounds (competitors). Data was fitted to a one-site binding competition non-linear regression curve with GraphPad Prism software using the following equation: Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}(X-LogEC50))

(28) FIG. 1 shows the results for competition with T4 for the binding to TTR wild type of competitors: Thyroxine (T4), Tolcapone (SOM), Tafamidis (TAF), and (−)-epigallocatechin-3-gallate (EGCG). The results are shown as the the curves of T4 displacement from TTR WT by the different compounds. From each dose-response curve, the EC.sub.50 value (inhibitor concentration at which half of the bound T4 is displaced) for each compound is determined. Further, the relative potency for the inhibition of binding of T4, defined as the ratio EC.sub.50 (T4)/EC50 (tested compound), was also calculated and is shown in table 3.

(29) TABLE-US-00003 TABLE 3 EC.sub.50 and relative potency of drug inhibition of T4 binding Relative potency of drug EC50 nM inhibition of T4 binding Thyroxine (T4) 50.11 nM 1 Tolcapone 41.85 nM 1.19 Tafamidis 214.4 nM 0.23 EGCG — No affinity

(30) These results demonstrate that tolcapone and tafamidis present similar binding affinity to TTR, while EGCG does not compete with T4 for the binding to TTR. The EC.sub.50 of tolcapone was 4 times lower than that of tafamidis, which demonstrates that tolcapone is more effective in binding the TTR tetramer, suggesting a higher anti-amyloidogenic potential.

Example 4: Assessment of TTR Tetrameric Stability by Isoelectric Focusing (IEF)

(31) To evaluate the effect of the tested compounds on TTR tetramer resistance to dissociation, TTR stability was assessed by IEF in semi-denaturing conditions as previously described (Ferreira et al, 2009, FEBS Lett, vol. 583, p. 3569-76). Samples were prepared as follows: 30 μL of human plasma from controls and TTR V30M carriers were incubated with 5 μl of 10 mM solution of test compounds and control (EGCG) compounds overnight at 4° C. followed by a 1 h incubation at RT. The preparations were subjected to native PAGE (5% acrylamide) and the gel band containing TTR was excised and applied to an IEF gel (5% acrylamide). IEF was carried out in semi-denaturing conditions (4 M urea), containing 5% (v/v) ampholytes pH 4-6.5 (GE Healthcare), at 1200 V for 6 hours. Proteins were stained with Coomassie Blue, the gels were scanned and subjected to densitometry using the ImageQuant program (HP Scanjet 4470c, Hewlett Packard). In the absence of any compound, plasma TTR presented a characteristic band pattern, composed of monomer, an oxidized monomer and several lower isoelectric point (pi) bands corresponding to different forms of tetramers. A total of 12 plasma samples (5 controls and 7 carriers TTR V30M) were analyzed in 3 IEF gels. For each treatment condition, a minimum of 4 samples from different donors were processed. The ratio of TTR tetramer over Total TTR (TTR tetramer+monomer) was calculated for each plasma sample and represented in FIG. 2. This ratio is normally higher for plasma from normal individuals than for the plasma from heterozygotic TTR V30M carriers plasma, as observed in FIG. 2. Treatment with tolcapone increases the amount of TTR tetramer over the monomeric forms compared to the non treated control plasmas of both normal or mutant TTR; and to a higher extent than tafamidis.

(32) The increase of the tetramer/total TTR ratio induced by the treatment with test compounds was pooled for all samples and represented in Table 4 as % of stabilization. These values were calculated after normalizing the tetramer/total TTR ratio obtained for each sample, with the ratio obtained for the non-treated plasma of the corresponding individual donor as described below: % stabilization=100×((ratio sample−ratio nt)/ratio nt). Where “ratio sample” is tetramer/total TTR ratio in the presence of compound; and “ratio nt” is tetramer/total TTR ratio of non-treated plasma from same donor.

(33) TABLE-US-00004 TABLE 4 Stability of TTR tetramer in the presence of compounds % stabilization (average +/− sem) Tolcapone 29.9 +/− 7.64 Tafamidis 16.4 +/− 5.49 EGCG 51.26 +/− 14.21

(34) Treatment with a TTR stabilizer such as tafamidis or tolcapone increases the ratio of tetramer over the monomeric forms. The results shown above clearly demonstrate that tolcapone presents a better stabilization effect on TTR tetramers than tafamidis.

Example 5: Cell Toxicity Assays

(35) To evaluate TTR-induced cytotoxicity and the preventive effect of the tested compounds, Rat Schwannoma cells (RN22, obtained from American Type Cell Collection ATCC), 80% confluent cells in Dulbecco's minimal essential medium with 10% fetal bovine serum, were exposed for 24 hours to 2 μM of TTR Y78F oligomers. These oligomers were obtained by incubation of soluble TTR Y78F either in the absence or presence of a 10× molar excess (final concentration is 20 μM) of test compounds or control (EGCG) at 37° C. for 6 days. Then, cells were trypsinized and cell lysates were used for determination of caspase-3 activation with the CaspACE fluorimetric 96-well plate assay system (Sigma). Protein concentration in lysates was determined with the Bio-Rad protein assay kit.

(36) The results obtained for caspase 3 activity and protein quantification in each cell culture well are represented in FIG. 3. Extracellular addition of non-treated TTR Y78F oligomers (control, O1) increased intracellular levels of Caspase-3, and thus cell death. TTR Y78F oligomers obtained in the presence of compounds that inhibit the formation of toxic oligomeric species (O2-O4) caused lower levels of Caspase-3 activation in RN22 cells. The reduction of cell toxicity in the presence of compounds (expressed as 100-% relative to control O1) is shown in table 5. It can be observed that tolcapone showed a greater reduction of cell cytotoxicity (29%) as compared to tafamidis (12%).

(37) TABLE-US-00005 TABLE 5 Reduction of cell toxicity in the presence of compounds Tolcapone 29% Tafamidis 12% EGCG 50%

Example 6: Fibril Disruption

(38) To study the effect of the test compounds on TTR fibrils disruption, we used TTR pre-formed fibrils prepared by incubation of a filtered (0.2 μm filters) solution of TTR L55P (2 mg/ml in PBS≈3.6 μM) for 15 days at 37° C. Subsequently, the samples were incubated either in the absence (control) or presence of a 10× molar excess (36 μM) (final concentration) of the test compounds for 4 days at 37° C. The disruption effect was evaluated by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) as previously described (Ferreira et al, 2009, FEBS Lett, vol. 583, p. 3569-76).

(39) It was observed that the control sample of TTR pre-formed fibrils (control) is mainly composed by big aggregates and fibrils (particles with a diameter higher than 1000 nm) and just a small amount of the protein is in soluble form (particles of 10 nm diameter). As the fibrils are being disrupted by the tested compounds the relative amount of big aggregates decrease and the small aggregates and soluble protein increase (see FIG. 4).

(40) The fibril disruption activity was quantified from the DLS analysis as the relative intensity (%) of aggregates and soluble particles after 4 days treatment with 36 μM of compounds (table 6).

(41) TABLE-US-00006 TABLE 6 DLS Analysis of TTR fibrils relative intensity (%) Soluble particles Aggregates Aggregates (~10 nm) (~10-100 nm) (~1000 nm) Control 28.2 — 71.8 tocalpone 56.1 5.9 38 Tafamidis 35.2 6.7 58.1 EGCG 49.1 26.3 24.6

(42) It can be observed that samples treated with tolcapone resulted in a higher amount of small aggregates and soluble proteins, thus exhibiting an important disruption activity. The results also show that tolcapone has a higher fibril disruption activity than tafamidis.

(43) The results obtained by experiments 1-6 clearly demonstrate that tolcapone has a high inhibitory activity of the formation of TTR amyloid fibrils and such inhibitory activity is higher than tafamidis, which has been described for the treatment of FAP. Further, tolcapone can disrupt pre-formed TTR amyloid fibrils more effectively than tafamidis. Altogether, the results indicate that tolcapone can be effectively used as a a medicament for the treatment of all types of TTR-associated amyloidosis.