TTR (transthyretin) (EC50 = 2.7-3.2 μM)

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].

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 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].

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. 

---

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).

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.

[1]. Bulawa, C.E., et al., 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.
[2]. Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis. Amyloid. 2016 Jun;23(2):109-18.

Tafamidis is a member of the class of 1,3-benzoxazoles that is 1,3-benzoxazole-6-carboxylic acid in which the hydrogen at position 2 is replaced by a 3,5-dichlorophenyl group. Used (as its meglumine salt) for the amelioration of transthyretin-related hereditary amyloidosis. It has a role as a central nervous system drug. It is a member of 1,3-benzoxazoles, a monocarboxylic acid and a dichlorobenzene. It is a conjugate acid of a tafamidis(1-).

---

Tafamidis and tafamidis meglumine (FX-1006A) are benzoxazole derivatives developed by FoldRX. Tafamidis is structurally similar to diflusinal. Tafamidis was granted an EMA market authorisation on 16 November 2011 and FDA approval on 3 May 2019.

---

Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive, life-threatening disease characterized by the aggregation and deposition of amyloidogenic misfolded transthyretin (TTR) in the myocardium. The gradual accumulation of insoluble TTR amyloid fibrils can result in restrictive cardiomyopathy and heart failure. Tafamidis (Vyndaqel®; Vyndamax®), a TTR stabilizer, has been approved for use in the treatment of adults with ATTR-CM in several countries. Tafamidis stabilizes both wild-type and mutant TTR, inhibiting the formation of TTR amyloid fibrils. In the pivotal phase III ATTR-ACT trial, tafamidis significantly reduced all-cause mortality and frequency of cardiovascular-related hospitalizations relative to placebo in patients with ATTR-CM. In addition, tafamidis recipients experienced significantly less deterioration in 6-minute walk test distance and quality of life than placebo recipients over the 30-month treatment period. Treatment benefits were largely consistent between patients with wild-type TTR and patients with a variant TTR genotype. Tafamidis was generally well tolerated in patients with ATTR-CM and, with a safety profile similar to that of placebo, tafamidis is suitable for long-term use. Given that treatment for this condition has in the past been largely limited to symptom management, tafamidis constitutes a valuable disease-modifying therapy for patients with ATTR-CM. References: Am J Cardiovasc Drugs. 2021 Jan;21(1):113-121.

C14H7CL2NO3

308.1163

306.980286

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

63.33

O=C(C1=CC=C2N=C(C3=CC(Cl)=CC(Cl)=C3)OC2=C1)O

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.

---

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.

---

View More

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.

---

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