Safety and efficacy of long-term diflunisal administration in hereditary transthyretin (ATTR) amyloidosis

Background: A recent 2-year randomized controlled trial indicated that the transthyretin (TTR) tetramer stabilizer, diflunisal, inhibits polyneuropathy progression and preserves quality of life in hereditary ATTR amyloidosis. However, its long-term outcomes are unknown. Here, we report tolerance and efficacy of long-term diflunisal administration in hereditary ATTR amyloidosis.

Methods: Diflunisal was administered orally at 500?mg/day to 40 Japanese hereditary ATTR amyloidosis patents who were not candidates for liver transplantation. The observation period ranged from 2 to 116 months (mean?±?SD: 38.0?±?31.2 months).

Results: Diflunisal-related adverse events included deterioration of renal function and thrombocytopenia resulting in discontinuation of the drug in three patients. Orally administered diflunisal significantly increased serum TTR concentration (p?=?0.001) and stabilized TTR tetramer structure in each patient. Longitudinal analyses of data collected at baseline, 24 months, and after 24 months confirmed sustaining effects of diflunisal on both neurological and cardiac functions. Notably, ulnar compound muscle action potential amplitude, cardiac wall thickness, and ejection fraction were not deteriorated after 24 months of treatment.

Conclusions: Diflunisal was tolerated well by most hereditary ATTR amyloidosis patients, although renal function and blood cell counts must be carefully monitored. Clinical effects of diflunisal were sustained after 2 years of treatment.     

In vivo stabilization of mutant human transthyretin in transgenic mice

Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.     

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Abstract

Objective: Transthyretin (ATTR) amyloidosis is an under-recognized, progressive disease manifesting as cardiomyopathy and/or polyneuropathy. Diflunisal, a nonsteroidal anti-inflammatory drug (NSAID), has demonstrated transthyretin stabilization in vitro and slowing of polyneuropathy progression in the hereditary ATTR subtype (ATTRm). However, the use of diflunisal has only been described in a small cohort of patients with ATTR cardiac amyloidosis (CA). We hypothesized that selected patients with ATTR-CA, both hereditary and wild-type (ATTRwt), would tolerate diflunisal with limited adverse events.

Materials and methods: This is a retrospective, longitudinal study of 23 patients with ATTR-CA (10 ATTRm and 13 ATTRwt) diagnosed at the Cleveland Clinic from May 2007 to August 2017 who were treated with diflunisal. Patients were prescribed diflunisal, fully informed of the risks of side effects. Patient characteristics and subsequent adverse events were recorded.

Results: The duration of diflunisal therapy ranged from 1–89 months (median 15?months). Average eGFR at diflunisal initiation was 61.9?±?15.4?mL/min/m². Only one patient had a transient rise in Cr of 0.31?mg/dL. There were no clinically significant bleeding events, despite most of the patients being on anticoagulants or antiplatelet agents. Three of 23 patients (13%) withdrew treatment due to drug side effects (erosive gastritis, epigastric pain and decreased appetite). No patients died or were hospitalized for heart failure.

Conclusion: Diflunisal was well-tolerated in both the ATTRm- and ATTRwt-CA populations. Withdrawal due to side effects was related to gastrointestinal complaints, but most patients had no adverse events. Diflunisal can be safely used in a selected group of ATTR-CA patients with appropriate clinical, renal and hematologic monitoring.     

ELIMINATION OF DIFLUNISAL AS ITS ACYL GLUCURONIDE, PHENOLIC GLUCURONIDE AND SULFATE CONJUGATES IN BILE-EXTERIORIZED AND INTACT RATS

1. The disposition of diflunisal (DF) was investigated in both bile-exteriorized and intact rats given 10 and 100 mg/kg doses intravenously (i.v.). 2. In addition to the phenolic glucuronide (DPG) and acyl glucuronide (DAG) conjugates, the sulfate conjugate (DS) was found to be a major metabolite. The glucuronides were excreted preferentially in bile, whereas DS was excreted almost exclusively in urine. 3. In bile-exteriorized animals, recoveries of DPG, DAG and DS in bile were 12.2%, 23.8%, 0.4%, respectively, and in urine, 10.3%, 5.6% and 15.2%, respectively, at the 10 mg/kg dose; and in bile, 11.3%, 41.6% and 1.0% respectively, and urine 2.9%, 1.1% and 17.0%, respectively, at the 100 mg/kg dose. 4. Total plasma clearance of DF and formation clearance of DF to DPG were reduced at the higher dose, suggesting saturation of this glucuronidation pathway. Formation clearances of DF to DAG and DS were little affected by the dose change. 5. Considerable enterohepatic recirculation of DF was apparent from the prolongation of DF and its conjugates in plasma of rats with an intact bile flow into the gut. The net metabolic effect of such cycling was enhancement of overall DS formation, from 15.6% and 18.0% of the 10 and 100 mg/kg doses, respectively, in bile-exteriorized rats to 28.5% and 42.1% of the doses respectively, in the intact animals.     

流动注射光度法测定二氟尼柳片的含量均匀度

在ｐＨ１的硝酸介质中，二氟尼柳可与Ｆｅ３＋生成紫色络合物，于５５０ｎｍ处有强吸收。基于上述原理，建立了流动注射光度法，并对二氟尼柳片的含量均匀度进行了测定，方法快速、简便。在０．０１２～０．１６８ｍｇ／ｍｌ浓度范围内线性关系良好，方法回收率为９５．７０％。     

Evaluation of the formalin test to assess the analgesic activity of diflunisal in the rat

The formalin test was evaluated to assess the analgesic activity of diflunisal in the rat. Fifty microliters of a 5% formalin solution was injected into the hindpaw of rats and two distinct nociceptive behaviors, i.e. flinching/shaking and licking/biting of the injected paw, were recorded over 120 min. The effect of factors such as age of the animal, time of injection (morning vs. afternoon), site of injection (right vs. left hind paw) were evaluated. Both nociceptive behaviors exhibited a biphasic time course. Rats weighing 210–220 grams showed a more intense response compared to older rats weighing 240–250 or 270–280 grams. The nociceptive behavior response was affected by the time of formalin injection and was more pronounced in the morning. Diflunisal (100 mg/kg, i.v. infusion over 3 min) caused a significant delay in the flinching/shaking response vs. time curve, whereas the licking/biting response was significantly inhibited. When carried out under carefully controlled conditions, the formalin test may be useful to study the analgesic effect of diflunisal in the rat. It seems to be less sensitive, however, than other commonly used nociceptive tests.     

In vitro drug interaction between diflunisal and indomethacin via glucuronidation in humans

It was reported that the plasma concentration of indomethacin was increased with concomitant oral dosages of diflunisal in humans. Both indomethacin and diflunisal are glucuronidated in humans. The effects of diflunisal on the indomethacin glucuronidation were thus investigated in vitro using human liver microsomes (HLM) and human intestine microsomes (HIM) in order to assess the drug-drug interaction. The glucuronidation of indomethacin in HLM showed atypical kinetics with Km and Ksi values of 210 and 89.5 microM, respectively, while HIM exhibited Michaelis-Menten kinetics with a Km value of 17.4 microM. Diflunisal inhibited the indomethacin glucuronidation in HLM with IC50 values ranging from 100 to 231 microM. In HIM, inhibition of the indomethacin glucuronidation by diflunisal was more potent with IC50 values of 15.2-48.7 microM. When the clinical dose of diflunisal (250 mg b.i.d.) is taken into consideration, it is expected that the diflunisal concentration in the intestine would be higher than the IC50 values for indomethacin glucuronidation in the intestine. These findings suggest that the clinical drug-drug interaction between diflunisal and indomethacin may be at least partly attributable to the inhibition of indomethacin glucuronidation by diflunisal in the intestine.     

二氟尼柳口服干凝胶剂的研制

以羧甲淀偻钠、低取代羟丙纤维素、甘露醇等为辅料,制备二氟尼柳口服干凝胶。实验表明,该制剂质量可控。采用紫外分不光度法测定其含量,线性范围８．０～７２．０μｇ／ｍｌ平均回收率１００．３１,ＲＳＤ为０．２６％。     

Sex-difference and the effects of smoking and oral contraceptive steroids on the kinetics of diflunisal

Summary The single dose pharmacokinetics of diflunisal were studied in 4 groups of 6 young volunteers: control men, control women, women taking low estrogen oral contraceptive steroids (OCS), and women smokers (10–20 cigarettes/day).The plasma clearance of diflunisal was significantly higher in men (0.169 ml·min^–1·kg^–1) and in women on OCS (0.165 ml·min^–1·kg^–1) as compared to control women (0.108 ml·min^–1·kg^–1). Partial metabolic clearances of diflunisal by the three conjugative pathways (phenolic and acyl glucuronide formation, sulphate conjugation) were all increased in men and women OCS users as compared to control women. Statistically significant increases, however, were only observed for the partial metabolic clearance of diflunisal by phenolic glucuronidation between men and women (2.91 vs. 1.85 ml·min^–1 respectively), and for the partial clearance by acyl glucuronidation between OCS users and control women (4.81 vs. 3.01 ml·min^–1 respectively).Smoking resulted in a moderate increase (35%) in plasma diflunisal clearance. However, a significant reduction in total urinary recovery of diflunisal and its glucuronide and sulphate conjugates was found in smokers (70.5% in smokers as compared to 84.2–87.2% in the 3 other study groups). Consequently, smoking may have induced hydroxylation, a minor oxidative metabolic pathway of diflunisal recently discovered in man.     

Falsely elevated salicylate levels due to diflunisal overdose

A 33-year-old man ingested 14 g of diflunisal (Dolobid^®, a NSAID), 3 propoxyphene-acetaminophen tablets, and ethanol. Although salicylate ingestion was specifically denied, toxicology screening revealed markedly elevated salicylate levels (176 mg/dL, 100 mg/dL, 41 mg/dL at 41/2, 10 and 24 hours postingestion). Blood ethanol level was 450 mg/dL. There was no respiratory alkalosis, metabolic acidosis, or increased anion gap to suggest salicylate toxicity. The patient recovered fully. Comparison testing of the salicylate assay technique used in the case (TDx^® fluorescence polarization immunoassay) and another commonly used technique (Trinder colorimetric assay) revealed marked cross-reactivity between salicylate and diflunisal. Samples of known diflunisal concentration were tested using the TDx^® salicylate assay. Percent cross-reactivity [(reported salicylate concentration/actual diflunisal concentration) × 100] was found to be 130 to 152.8% over the concentration range tested. Cross-reactivity between diflunisal and salicylate using the Trinder colorimetric assay was 20 to 52.5010 over the concentration range tested. Diflunisal is not metabolized to salicylate and thus this cross-reactivity rearesents a significant drug-laboratory interaction. Salicylate levels measured by these methods appear to be unreliable when diflunisal is present.