The goals of the treatment of hypothyroidism are “to achieve a state of euthyroidism and normalization of the circulating levels of TSH and thyroid hormones” (1), whereby TSH is used as a powerful and reliable proxy for euthyroidism owing to its robust correlation with free T4 concentration (2). This is commonly achieved with levothyroxine (LT4) which is relatively inexpensive, available in multiple strengths and administered in single dose. This strategy relies on the conversion of the prodrug LT4 into its active metabolite T3 to correct the lack of endogenous T3 production (estimated as 15% of the total circulating pool) from the thyroid gland (3). Experimental data (4, 5) indicate that LT4 alone is not sufficient to restore tissue euthyroidism, and clinical observations (6) demonstrate that circulating levels of T3 are reduced in patients receiving LT4 therapy. Clinically, a significant percentage of patients adequately treated with LT4 complain of residual symptoms which may be attributed to hypothyroidism (7). Collectively, these observations have prompted interest in LT4/Liothyronine (synthetic T3, LT3) combination therapy or desiccated thyroid extracts as means to correct for the loss of endogenous T3, and improve symptomatology (8).

Whereas most clinical studies have showed modest to nihil improvement in symptoms and quality of life (1), a plurality of patients appeared to prefer combination therapy (9, 10). Moreover, a secondary analysis of the largest study indicated that carriers of the Ala92 allele of the type 2 deiodinase gene showed a significant improvement in quality of life (11). These observations contribute to a renewed interest in the therapeutic and pharmacologic use of LT3 (8, 12, 13), with a specific interest in developing “new well-designed adequately powered clinical trial of combination therapy” (14).

The pharmacokinetic characteristics of LT4 allow for once-daily administration. In contrast, LT3 has a short distribution half-life resulting in significant changes in serum T3 concentrations (15). Thus, a once-daily administration regimen would result in post-absorptive peaks above the normal range (15). Since extended release formulations of LT3 are not available, in LT3/LT4 combination therapy a twice daily low-dose LT3 administration is recommended to minimize fluctuations of serum T3 (13, 14). The tradeoff is a cumbersome regimen which is not convenient for a lifelong therapy, as well complicating the recruitment and retention in clinical studies (16).

The main concern for cardiovascular complications due to transient rise in serum T3 concentrations above the normal range stems from extrapolations from chronic exposure to supraphysiologic doses (either endogenous or exogenous) of T3, and from the in vitro evidence of rapid, non-genomic effects of T3 in vascular endothelium and cardiomyocytes (17, 18), which would be directly exposed to fluctuations in the T3 concentrations as a result of LT3 administration. We hypothesized that if there is a clinically relevant rapid action of LT3, we would be able to detect measurable and clinically-significant, temporal changes in cardiovascular function and energy expenditure following a high, single-dose LT3 ( Figure 1 ). Otherwise, from the clinical perspective, the action of LT3 would be entirely attributable to long-term transcriptional effects, and transient changes in serum T3 concentrations would not be a concern, supporting single daily administration of LT3 alone or in combination with LT4.

Here we present a pharmacokinetics-pharmacodynamics study designed to assess the clinical relevance of rapid effects of T3, with a particular focus on myocardial function, systemic vascular resistance, and energy metabolism.

LT3 therapy for hypothyroidism is associated with weight loss, decreased cholesterol concentrations and a trend toward improved diastolic function (10). Very recently, similar findings on weight and lipid parameters have been reported in elderly patients affected by subclinical hypothyroidism (11). Additionally, experimental evidences suggest that low dose supplementation of LT3 improves myocardial contractility in patients with congestive heart failure (12). Moreover, a secondary analysis of the largest LT3/LT4 combination therapy study (13) showed improved quality of life in carriers of the inactivating Thr92Ala type-2 deiodinase polymorphism receiving LT3 supplements (14). Of interest, in this trial LT3 was administered in single daily dose. Since LT3 was originally approved for treatment of hypothyroidism in 1956 (15), this drug did not undergo the rigorous clinical testing currently necessary for approval and has been “grandfathered” for current use. Although the package insert recommends once-a day administration, the consensus among thyroidologists is to subdivide the dose to limit the fluctuations in serum T3 concentrations (16). Because of lack of long-term studies on its safety and effectiveness, the American Thyroid Association recommends against its use for the treatment of hypothyroidism (17). Specifically, the concerns for potential toxicity of supraphysiologic serum concentrations of T3 has hampered the use of LT3 alone or LT3/LT4 combination therapy in patients with hypothyroidism.

LT3 is promptly absorbed following oral administration, and doses as low as 0.25 mcg/Kg result in increase in T3 serum concentration well above the upper limit of reference, raising the concern for cardiovascular toxicity. Rapid, non-genomic actions of thyroid hormone have been demonstrated in vitro in endothelial vascular cells (18), providing additional rationale for the concern for toxicity due to the exposure of the vasculature to transient increase in T3 serum concentrations following LT3 administration. The interaction between T3 and integrins results in the production of nitric oxide, promoting vasodilation (19, 20). Despite these laboratory-based data, the clinical relevance of rapid effects of thyroid hormone is not clear. Studies performed in ex vivo models of vasculature contractility are conflicting: Gachkar and colleagues demonstrated a rapid response to physiologic concentrations of T3, and a decreased response to both hyper- and hypothyroid concentrations, not mediated by AKT, ERK or AMPK (21). More recently, other authors demonstrated an increase in vasodilation following exposure to T3, mediated by PI3K pathway and thyroid hormone receptor alpha (22). Experiments conducted in an animal model of hypothyroidism indicate that exposure to high dose LT3 generates a measurable transcriptional effect within 30’ reaching a maximum effect after 6 hours (23). Clinically, the reports of toxicity secondary to trauma with release of thyroid hormone from the gland, or following acute poisoning indicate a significant lag time (24, 25), suggesting that the majority of the harm caused by thyrotoxicosis is ascribable to transcriptional effects of thyroid hormone. It is possible that the discrepancies between laboratory-based experiments and in vivo observations could be explained by the dynamic of acute exposure (seconds) to thyroid hormone vs. the much slower changes in thyroid hormone concentrations following oral administration.

We speculated that, if rapid effects of T3 were of clinical relevance, transient exposure to T3 concentrations above normal range would result in measurable and potentially clinically significant changes in the cardiovascular system and energy metabolism owing to the exquisite sensitivity of these end-organ targets to the action of thyroid hormone (26, 27). Moreover, since the endothelial vascular cells are exposed to changes in T3 concentrations, measurement of their function by assessing systemic vascular resistance would represent an ideal readout to assess acute effects of supraphysiologic doses of LT3.

To test this hypothesis, we conducted a pharmacokinetics/pharmacodynamic study directed to these targets of the hormonal action. In this study we chose to investigate a short timeframe based on our knowledge of liothyronine PK (1), aiming to capture events occurring at and immediately after the C_max, i.e. at the time of maximal exposure of endothelial and myocardial cells to the peak serum T3 concentrations. We used a pharmacologic dose which would produce peak T3 concentration well above the upper reference range. To control for potential non-specificity in the nongenomic effects of thyroid hormone, in addition to placebo we included a study arm of equimolar LT4 dose as additional control.

By using liquid formulations of LT3 and LT4 we were able to titrate the doses to adjust for the participants’ weight, further increasing the internal validity of the study. When compared to our previous observations on tablet formulation (1), liquid LT3 showed a similar T_max, while, as expected, the C_max was much greater due to the higher LT3 dose employed in this study. The decrease in TSH from baseline to the end of the study, which is consistent with the hormone biorhythm (28) and possibly amplified by the prolonged fast, was not different among the active drugs and placebo.

Our data indicate no significant differences or appreciable trend during our observation in the all the study endpoints despite achieving clearly supraphysiologic serum T3 concentrations. Specifically, during the four hours following the administration of LT3, we did not observe any change in HR or BP, or estimates of CO and SVR. Moreover, we independently assessed the acute effects of LT3 on the myocardium by performing a Doppler echocardiography study before and after each of the studies. Similar to the ccNexfin continuous recordings, we observed no measurable effects on myocardial contractility. Finally, no differences were observed in energy metabolism measured by the WRIC.

Our data are consistent with the observations of Jonklaas et al. that showed significant increase in heart rate and blood pressure only after five hours from the administration of a pharmacologic dose of LT3 (29). Of note, in their study, the subjects were given lunch at 4-hour, which per se may have resulted in post-prandial HR elevation (30); thus, the true cardiovascular response beyond 4 hours without confounding factors remains to be captured.

Collectively, the results of our study indicate that a single administration of LT3, sufficient to transiently increase the T3 concentration well above the range of reference, does not result in significant signal in the tissues which are most sensitive to acute effects of T3, and are immediately exposed to the peak in T3 concentration. These findings appear to negate clinical relevance to the rapid effects of T3 which have been observed in vitro. The clinical implications are intriguing, since one could speculate that these results provide the rationale to consider the use of single administration of LT3, rather than trying to reach stable serum concentrations by extended release formulations or by multiple daily administrations regimens. Conversely, we want to affirm that absent an empirical demonstration of effectiveness and safety of once daily administration regimen, this interpretation of the data should not translate in clinical practice.

Strengths of our study reside in the rigorous study design, use of liquid formulation of the drug which allowed for a precise, weight-based dosing, and the use of state-of-the-art techniques to assess the cardiovascular system and energy metabolism. The continuous recording of BP, endothelial vascular function and EE allowed us to measure even subtle changes which would not be captured by single timepoint observations. Finally, we included a treatment arm of LT4 to assess for potential non ligand-specific acute effects of thyroid hormone. To the best of our knowledge, no study has investigated the rapid effects of thyroid hormone in such details.

The relatively small sample size, non-unusual in phase I-II studies, represents a limitation. However, the absence of trends in the physiological variables indicates that it is extremely unlikely our negative findings are due to type-2 error. Some volunteers had a short, 48-hour interval between the studies raising the potential concern for carryover effects. Given the lack of measurable effects and intraindividual differences of baseline (before administration of the study drugs) data, we do not believe that this is a cofounding factor. By design, the observation was limited to a timeframe where maximum variation in serum T3 concentrations can be observed, therefore late events would not be captured. Lastly, the translational value of observation obtained in healthy individuals to patients affected by hypothyroidism, in particular elderly and with co-morbidities is unknown, and we cannot rule out the possibility that acute effects may occur in patients with lower serum T3 levels.

In conclusion, in healthy individuals a single administration of LT3, able to rapidly increase the serum T3 concentration above normal range does not result in measurable changes in target organ-systems attributable to the rapid thyroid hormone action. The data support the rationale to explore the use of LT3 in single dose aiming to achieve sustained increase in tissue concentrations of T3, reducing the concern for fluctuations in serum concentrations. On the other hand, patients who have longstanding significant hypothyroidism may experience paradoxical response, and T3-based therapy should be started after achieving a euthyroid state by LT4, and residual symptoms are still present. Long-term studies in patients affected by hypothyroidism are necessary to assess the safety and effectiveness of single dose LT3 or LT3/LT4 combination therapy in patients affected by hypothyroidism.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by VCU IRB. The patients/participants provided their written informed consent to participate in this study.

SCh contributed to the study design, analyzed energy expenditure data, performed statistical analysis and contributed to the interpretation, and contributed to the initial draft and final writing of the manuscript. GFW performed pharmacokinetics analysis and contributed to the writing of the manuscript. AV recorded and analyzed echocardiography data. SCa recorded and analyzed hemodynamics data. SY contributed to the study design, clinical assessment of study patients, and contributed to the editing of the manuscript. BVT contributed to the study design and interpretation of the pharmacokinetics data. AA contributed to the study design and interpretation of the echocardiography data. FSC designed the study, supervised the analysis and interpretation of the data, and wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.

SCa is supported by a Career Development Award 19CDA34660318 from the American Heart Association and by the Clinical and Translational Science Awards Program UL1TR002649 from National Institutes of Health to Virginia Commonwealth University. FSC is supported by the NIH-NIDDK grant 1 R21 DK122310-01A1.

Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

The Division of Endocrinology, Diabetes and Metabolism of Virginia Commonwealth University has received an unrestricted grant from IBSA Institut Biochimique, Lugano Switzerland. Liquid formulations of LT3, LT4, and placebo were a kind gift of IBSA Institut Biochimique Lugano (Switzerland). IBSA was not part of the study design and had no access to the data prior to the publication. FC has served as consultant for IBSA, Acella and Kashiv (now merged with Amneal).

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.