TTR (transthyretin) (EC50 = 2.7-3.2 μM)
Tafamidis meglumine targets the transthyretin (TTR) protein, a tetrameric serum and cerebrospinal fluid transporter of thyroxine and retinol-binding protein. TTR tetramer dissociation into monomers is the rate-limiting step in amyloidogenesis, and the drug binds selectively to the two common polarin sites of the TTR tetramer with negative cooperativity, exhibiting dissociation constants (Kds) of approximately 2 nM and 200 nM. By stabilizing the native tetrameric conformation, it prevents the misfolding and aggregation of TTR monomers into amyloid fibrils, thereby halting the progression of TTR-related amyloidosis.

Tafamidis kinetically stabilizes TTR by inertly binding the two common polarin sites of the tetramer with negative affinity (Kds ∼2 nM and ∼200 nM) [1]. After 72 hours at pH 4.4-4.5, tacramamide (0-7.2 μM) quantitatively suppresses WT-TTR amyloidosis [1].
In vitro, tafamidis kinetically stabilizes TTR by binding inertly to the two common polarin sites of the tetramer with negative cooperativity. It shows comparable potency and efficacy across wild-type TTR (WT-TTR) and mutant homotetramers including V30M-TTR and V122I-TTR, with EC50 values ranging from 2.7 to 3.2 μM. After 72 hours of incubation at pH 4.4-4.5, tafamidis at concentrations of 0-7.2 μM quantitatively suppresses WT-TTR amyloidogenesis. This demonstrates its ability to prevent the acid-mediated dissociation of the TTR tetramer and subsequent amyloid fibril formation in a cell-free system.

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis[2].
In vivo, ATTR amyloidosis is a systemic, debilitating, and fatal disease caused by the accumulation of TTR amyloid deposits. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNA interference (RNAi) technology has been evaluated in murine models of hereditary ATTR amyloidosis to achieve robust hepatic gene silencing, with the goal of reducing systemic TTR levels and mitigating the clinical manifestations of ATTR that arise from hepatic TTR expression. While RNAi represents a promising therapeutic approach, tafamidis itself has demonstrated clinical efficacy in slowing disease progression in patients with FAP.

Immunoturbidity Assay for Stabilization of TTR Tetramer in Human Plasma.[1] Urea denaturation of TTR in human plasma and chemical crosslinking was performed as described (see text and Fig. 6.) with minor modifications, except that TTR was quantified by immunoturbidity. Human plasma samples were thawed on ice and insoluble material was removed by centrifugation. For each, 4 µL was removed, and the initial TTR concentrations were determined by immunoturbidity. For each stabilization determination, 80 µL aliquots of each plasma sample were retained and 1.6 µL of either 5% dimethyl sulfoxide (DMSO) or 360 µM tafamidis in 5% DMSO was added. After incubation at room temperature for 15 minutes, 120 µL of urea buffer (8 M urea, 40 mM sodium phosphate, 80 mM KCl, pH 7.4) was added and samples were mixed and incubated at room temperature for the indicated time (typically 48 h). All samples were cross-linked with 3.2 µL of 25% glutaraldehyde. After 4 minutes, the reaction was quenched with 5.6 µL of 1.85 M NaBH4 (freshly prepared in 0.1 N NaOH) and incubated for 5 minutes. Postdenaturation TTR concentrations (4 µL) were determined by immunoturbidity. Olympus OSR6175 reagent and Prealbumin Calibrator ODR3029 were used according to the manufacturers’ instructions. To assess the correlation between the two detection methods, we analyzed plasma samples after urea treatment and glutaraldehyde crosslinking in parallel by Western blot and immunoturbidity. In the control samples, the amount of TTR detected by immunoturbidity decreased from an initial value of 22 mg/dL to 3 mg/dL after 3 d in urea. In the presence of tafamidis, 13 mg/dL of TTR remained; a level that was in good agreement with results from the Western blot assay (Fig. S3A).
The in vitro enzyme/receptor binding (non-cellular) assay for tafamidis meglumine typically involves a fluorescence-based kinetic stabilization assay using purified recombinant TTR protein. The assay measures the ability of the compound to prevent acid-mediated dissociation of the TTR tetramer. TTR is incubated with varying concentrations of tafamidis (typically 0-7.2 μM) at pH 4.4-4.5 for 72 hours. The extent of amyloid fibril formation is then quantified using a fluorescent dye such as thioflavin T, which exhibits enhanced fluorescence upon binding to amyloid fibrils. The concentration required to achieve 50% inhibition of amyloidogenesis (EC50) is determined from the dose-response curve.

In vitro cellular assays for tafamidis meglumine are limited because TTR is primarily a serum protein and its amyloidogenesis is typically studied in cell-free systems. However, cellular models can be employed to assess the downstream effects of TTR stabilization. For example, cell lines expressing mutant TTR can be treated with tafamidis, and the levels of secreted TTR and its aggregation state can be monitored. Additionally, cytotoxicity assays can be performed to evaluate the protective effects of tafamidis against TTR aggregate-induced cell death in neuronal or other relevant cell types. The compound's ability to reduce extracellular TTR amyloid deposition in co-culture systems may also be examined.

Evaluation of tafamidis in hTTR V30M HSF1± mice[2] Tafamidis/meglumine (tafamidis) and its respective meglumine only control (meglumine) were prepared as previously described. Four hundred microliters of 2 mg/ml tafamidis (0.8 mg total) or its respective meglumine control were administered via subcutaneous injection to 15-month-old hTTR V30M HSF1± mice on days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, 31, 33, 35 and 38. TTR tissue deposition was evaluated on day 52 as described earlier. To confirm tafamidis-mediated stabilization of serum TTR, serum TTR tetramer stability was analyzed on days -7, 9, 23 and 37 using a modified version of a previously described TTR tetramer stability assay. See Supplementary Figure 2 for more detail on assay conditions and tetramer detection and quantitation. To quantify the extent of stabilization, % TTR tetramer stabilization was calculated using the following equation as previously described.
In vivo animal studies for tafamidis meglumine have been conducted in murine models of hereditary ATTR amyloidosis. In these models, mice transgenic for human mutant TTR are administered tafamidis orally, and the effects on TTR tetramer stabilization, amyloid deposition in tissues, and disease progression are evaluated. Additionally, RNAi-mediated silencing of hepatic TTR has been assessed as a therapeutic strategy. Pharmacodynamic endpoints include measurement of plasma TTR levels, assessment of TTR tetramer stability ex vivo, and histopathological examination of tissue amyloid deposits. The drug's ability to improve survival and clinical parameters is also evaluated.

Tafamidis meglumine is orally bioavailable. As a meglumine salt of tafamidis, it is formulated for oral administration. The drug is absorbed and distributed systemically, reaching therapeutic concentrations in the plasma to stabilize circulating TTR. The pharmacokinetic profile of tafamidis supports once-daily dosing, as the drug has a sufficiently long half-life to maintain sustained TTR stabilization throughout the dosing interval. Detailed pharmacokinetic parameters such as Cmax, Tmax, AUC, and half-life are typically derived from clinical studies in healthy volunteers and patients with ATTR amyloidosis. The drug is primarily metabolized in the liver and excreted via the biliary route.

Tafamidis meglumine is generally well-tolerated. The most common adverse events reported in clinical trials include mild to moderate gastrointestinal disturbances, urinary tract infections, and headache. Serious adverse events are rare. The drug has a favorable safety profile, with no significant hepatotoxicity or nephrotoxicity observed in preclinical or clinical studies. Long-term safety data from post-marketing surveillance continue to support its use as a chronic therapy for ATTR amyloidosis. As a TTR stabilizer, tafamidis does not exhibit the off-target toxicity associated with many small molecule drugs, primarily due to its high selectivity for the TTR binding site.

[1]. Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade. Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9629-34.

Tafamidis meglumine is an organic ammonium salt composed of Tafamidis and an equimolar amount of 1-deoxy-1-(methylamino)-D-glucol. It is used to improve transthyretin-associated hereditary amyloidosis. It is a central nervous system drug. It contains a Tafamidis (1-) group. Fx-1006A is a small molecule compound with the potential to treat hereditary diseases such as familial amyloid cardiomyopathy (FAC) and familial amyloid polyneuropathy (FAP). Tafamidis meglumine is a soft capsule formulation containing the meglumine salt form of Tafamidis, a small molecule pharmacological chaperone with potential disease-modifying activity. Tafamidis binds to and stabilizes the structure of wild-type and variant (V122I) transthyretin (TTR), preventing the tetramer from dissociating into monomers; this prevents TTR protein misfolding, inhibits the formation of TTR amyloid fibrils, and the subsequent deposition of these insoluble protein clusters in peripheral nerve tissues and organs. TTR is a 127-amino acid thyroxine and retinol transporter secreted by the liver.
See also: Tafamidis (with active moiety).
Pharmaceutical Indications

Studied for the treatment of amyloidosis.
Neurogenic Hereditary Amyloidosis

Mechanism of Action
Fx-1006A is a first-in-class disease-modifying compound designed to inhibit the formation of amyloid deposits by preventing the misfolding and deposition of transthyretin (TTR) associated with amyloidosis.
Tafamidis meglumine is an FDA-approved and EMA-approved medication for the treatment of transthyretin-mediated amyloidosis (ATTR) in adults with cardiomyopathy (Vyndamax, Vyndaqel). It is the first and only approved therapy that directly targets TTR stabilization. The recommended dose for Vyndaqel (tafamidis meglumine) is four 20 mg capsules orally once daily (80 mg total), while Vyndamax (tafamidis free acid) is administered as one 61 mg capsule once daily. Clinical trials have demonstrated that tafamidis significantly reduces all-cause mortality and cardiovascular-related hospitalizations in patients with ATTR cardiomyopathy. The drug represents a major advance in the treatment of this previously untreatable disease.

C21H24CL2N2O8

503.329864501953

502.091

C, 50.11; H, 4.81; Cl, 14.09; N, 5.57; O, 25.43

951395-08-7

Tafamidis;594839-88-0

24970412

Typically exists as White to off-white solids at room temperature

1.532

7

10

8

33

506

4

[C@@H](O)([C@@H](O)CNC)[C@H](O)[C@H](O)CO.ClC1C=C(Cl)C=C(C2=NC3C=CC(=CC=3O2)C(=O)O)C=1

DQJDBUPLRMRBAB-WZTVWXICSA-N

InChI=1S/C14H7Cl2NO3.C7H17NO5/c15-9-3-8(4-10(16)6-9)13-17-11-2-1-7(14(18)19)5-12(11)20-131-8-2-4(10)6(12)7(13)5(11)3-9/h1-6H,(H,18,19)4-13H,2-3H2,1H3/t4-,5+,6+,7+/m.0/s1

(2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol 2-(3,5-dichlorophenyl)benzo[d]oxazole-6-carboxylic acid

Fx 1006A; PF-06291826; Fx-1006A; PF 06291826; Fx1006A PF06291826; Tafamidis meglumine; Vyndaqel; 951395-08-7; Fx-1006A; Fx1006A; Fx 1006A; UNII-ZU7CF08A1A; ZU7CF08A1A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~12.5 mg/mL (~24.83 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (2.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (2.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)