TTR (transthyretin) (EC50 = 2.7-3.2 μM)
Transthyretin (TTR) tetramer (binds with negative cooperativity to the two thyroxine-binding sites; \(K_{d1} \sim 2-3 \text{ nM}\), \(K_{d2} \sim 154-278 \text{ nM}\); EC\(_{50}\) ~2.7–3.2 µM for fibril inhibition under acidic conditions) [1]

TTR is kinetically stabilized when tacamimeis binds to the two tetramer's typically conserved polarin binding sites with negative coupling (Kd = ∼2 nM and ∼200 nM) [1]. After 72 hours at =4.4-4.5, tacramids (0-7.2 μM) dose-dependently suppresses WT-TTR amyloidosis [1].

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Tafamidis dose-dependently inhibits amyloid fibril formation by wild-type (WT), V30M, and V122I TTR homotetramers under acidic, fibril-promoting conditions (pH 4.4–4.5, 72 h incubation), with EC\(_{50}\) values corresponding to a tafamidis:TTR molar ratio of <1.
Tafamidis kinetically stabilizes TTR tetramers against urea-mediated denaturation (6.5 M urea, 72 h), reducing tetramer dissociation to less than 3% at a 2:1 molar ratio.
Tafamidis selectively binds to TTR in human plasma with a stoichiometry of ~0.81 ± 0.02 tafamidis per TTR tetramer, indicating high selectivity among plasma proteins.
Tafamidis stabilizes a broad range of pathogenic TTR variants (V30M, Y69H, F64S, I84S, V122I, L111M) in plasma under urea denaturation stress.[1]

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].

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In a phase II/III clinical trial of tafamidis in V30M TTR-FAP patients, this kinetic stabilizer demonstrated clinical efficacy over 18 mo of treatment. Relative to placebo controls, patients receiving tafamidis had 52% less neurologic deterioration, 53% and 80% preservation of large- and small-nerve fiber function, and improved nutritional status, outcomes that are associated with an improved quality of life. The tafamidis preclinical data presented within, when considered in concert with the clinical efficacy data, provide unique pharmacologic evidence supporting the amyloid hypothesis, the notion that lowering the efficiency of the amyloid cascade halts the degeneration of the peripheral and autonomic nervous system[1].

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In a Phase II/III clinical trial in V30M TTR-FAP patients, tafamidis treatment over 18 months resulted in 52% less neurological deterioration, 53–80% preservation of nerve fiber function, and improved nutritional status compared to placebo.[1]

Tafamidis Binds with High Affinity to TTR at Its T4-Binding Sites. Tafamidis Stabilizes the Weaker TTR Dimer–Dimer Interface.  Tafamidis Binds Selectively to TTR in Human Plasma. Tafamidis Stabilizes WT, V30M, and V122I TTR in Human Plasma. Tafamidis Stabilizes a Broad Range of Pathogenic TTR Variants. 

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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).

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Isothermal titration calorimetry (ITC) was used to determine binding constants. A solution of tafamidis was titrated into a cell containing WT-TTR (17 µM) at 25°C. Heat changes were measured and integrated to generate a binding isotherm, which was fit to a negative cooperativity model yielding \(K_{d1} = 3 \text{ nM}\) and \(K_{d2} = 278 \text{ nM}\).[1]
Subunit exchange experiments at physiologic pH were used to indirectly determine tetramer dissociation rates and calculate binding constants (\(K_{d1} = 2 \text{ nM}\), \(K_{d2} = 154 \text{ nM}\)) by analyzing the fraction of unbound TTR tetramers as a function of tafamidis concentration.[1]

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.
To compare the efficacy of the tetramer stabilization approach to that of RNAi-mediated TTR knockdown, we evaluated tafamidis in the hTTR V30M HSF1± model and quantified the impact of TTR tetramer stabilization on the regression of preexisting TTR deposits. To compensate for differences in dose frequency and route of administration, mice were administered excess tafamidis (>100× on mg/kg basis) to enable sufficient TTR tetramer stabilization. Although administration of tafamidis resulted in a significant and clinically relevant degree of serum TTR tetramer stabilization, only moderate TTR deposit regression was observed in the sciatic nerve and dorsal root ganglion; consistent regression was not observed in other tissues examined. It should be noted that the study duration was chosen to allow a more direct comparison with siTTR1 (Figure 3) and, as such, it is possible that longer term administration of tafamidis may have resulted in greater deposit regression in the hTTR V30M HSF1± model. However, in these conditions, TTR lowering seems to be more effective.

Absorption, Distribution and Excretion
The peak plasma concentration (Cmax) of tafamid was 1430.93 ng/mL, with a fasting time to peak concentration (Tmax) of 1.75 hours and a postprandial time to peak concentration (Tmax) of 4 hours. The AUC of tafamid was 47,864.31 ng/mL. After oral administration of 20 mg tafamid, approximately 59% is excreted in feces, primarily in its unchanged form. Approximately 22% is excreted in urine, primarily as glucuronide metabolites. The steady-state apparent volume of distribution was 18.5 L. The oral clearance of tafamid was 0.263 L/h. The apparent total clearance was 0.44 L/h. Metabolism/Metabolites In vitro studies showed that tafamid was largely unaffected by first-pass metabolism or oxidative metabolism, with 90% of its metabolites remaining unchanged. Preclinical data indicate that tafamid is primarily metabolized via glucuronidation and excreted via bile.

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Biological half-life
The half-life of tafamid is 49 hours.

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Tafamids has oral bioavailability and selectively binds to TTR in the blood, while its binding to other plasma proteins is not significant. [1]
The plasma half-life of TTR is approximately 24 hours, and subunit exchange assays have shown that tafamid can stabilize TTR during this period. [1]

Protein Binding
Tafamidis has a 99.9% protein binding rate in plasma, primarily binding to transthyretin. Tafamidis was well-tolerated in clinical trials; although it binds to the thyroxine binding site on the TTR, it has no clinically relevant effect on thyroid function or laboratory parameters. [1]

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

Tafamidis belongs to the 1,3-benzoxazole class of compounds and has the structure 1,3-benzoxazole-6-carboxylic acid, where the hydrogen at the 2-position is replaced by a 3,5-dichlorophenyl group. It (in the form of meglumine salt) is used to improve transthyretin-associated hereditary amyloidosis. It is a central nervous system drug. Tafamidis belongs to the 1,3-benzoxazole class of compounds and is a monocarboxylic acid and dichlorobenzene. It is the conjugate acid of tafamidis (1-). Tafamidis and tafamidis meglumine (FX-1006A) are benzoxazole derivatives developed by FoldRX. The structure of tafamidis is similar to diflusinal. Tafamidis received EMA marketing authorization on November 16, 2011, and FDA approval on May 3, 2019. See also: Tafamidis meglumine (salt form). Drug Indications Tafamidis is indicated for the treatment of wild-type or hereditary transthyretin-mediated amyloid cardiomyopathy in adults. FDA Label Mechanism of Action Genetic mutations or natural misfolding of transthyretin can disrupt the stability of the transthyretin tetramer, leading to its dissociation and aggregation in tissues, thereby disrupting the normal function of these tissues. Tafamidis binds to the transthyretin tetramer at the thyroxine binding site, stabilizing the tetramer and reducing the number of monomers available for amyloidosis. Pharmacodynamics Tafamidis stabilizes the transthyretin tetramer, reducing the number of monomers available for amyloidosis. Because it only requires once-daily dosing, it has a long duration of action and a wide therapeutic window.

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Tafamidis is a benzoxazole derivative (2-(3,5-dichlorophenyl)-benzoxazole-6-carboxylic acid) developed as an orally effective TTR kinetic stabilizer to inhibit tetramer dissociation, the rate-limiting step in the TTR amyloidosis process.
It mimics the protective effect of the T119M mutation, which slows tetramer dissociation and prevents complex heterozygote disease.
TTR-bound crystal structure (1.3 Å resolution) shows that it occupies the thyroxine binding site, and the hydrophobic interaction of the dichloro substituent and the water-mediated hydrogen bond formed through its carboxylic acid group stabilize the dimer-dimer interface. [1]

C14H7CL2NO3

308.1163

306.98

C, 54.58; H, 2.29; Cl, 23.01; N, 4.55; O, 15.58

594839-88-0

Tafamidis meglumine;951395-08-7;Tafamidis-d3

11001318

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

1.5±0.1 g/cm3

486.7±40.0 °C at 760 mmHg

248.1±27.3 °C

0.0±1.3 mmHg at 25°C

1.677

5.29

1

4

2

20

371

0

ClC1C([H])=C(C([H])=C(C=1[H])C1=NC2C([H])=C([H])C(C(=O)O[H])=C([H])C=2O1)Cl

TXEIIPDJKFWEEC-UHFFFAOYSA-N

InChI=1S/C14H7Cl2NO3/c15-9-3-8(4-10(16)6-9)13-17-11-2-1-7(14(18)19)5-12(11)20-13/h1-6H,(H,18,19)

2-(3,5-dichlorophenyl)benzo[d]oxazole-6-carboxylic acid

Fx-1006, PF06291826; Fx1006, PF-06291826; Fx 1006, Fx-1006A; PF 06291826; Tafamidis; Vyndaqel; TAFAMIDIS; 594839-88-0; Vyndamax; FX-1006; 2-(3,5-Dichlorophenyl)-1,3-Benzoxazole-6-Carboxylic Acid; 2-(3,5-Dichlorophenyl)-6-benzoxazole carboxylic acid; tafamidisum; 8FG9H9D31J;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~37.5 mg/mL (~121.71 mM)

Solubility in Formulation 1: 2.5 mg/mL (8.11 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: 2.5 mg/mL (8.11 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (8.11 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 25.0 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.)