Hypothyroidism Express Report


Issues in Determining Bioequivalence in Levothyroxine Sodium Formulations

This report was reviewed for medical and scientific accuracy by Stephen H. Schneider, MD, Professor of Medicine, Division of Endocrinology, Metabolism and Nutrition,University of Medicine & Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey


Determination of bioequivalence between levothyroxine sodium formulations is made challenging by two inherent characteristics of levothyroxine. First, exogenous levothyroxine sodium is biochemically and physiologically indistinguishable from endogenously produced thyroxine, whose production is controlled by the hypothalamic-pituitary-thyroid axis.1 Second, levothyroxine sodium has a narrow therapeutic range.2 A narrow therapeutic range is defined as the narrow difference between plasma levels that produce desired pharmacologic effects and levels that can induce toxicity. Therefore, safe and effective use of levothyroxine sodium requires careful titration and clinical monitoring to avoid putting patients at risk for iatrogenic hyper- or hypothyroidism. Such risk can occur at doses only 25% less or greater than optimal, based on patients' serum thyroid-stimulating hormone (TSH) concentration.3

It is for these reasons that the American Thyroid Association (ATA) and the American Association of Clinical Endocrinologists (AACE) recommend that physicians use a single brand of levothyroxine sodium longitudinally, without crossover between brands, in order to safeguard patients requiring thyroid hormone therapy from excessive or insufficient doses of hormone.4,5 Data from investigations6,7 and studies of clinical practices8 suggest that 15% to 29% of patients currently receive inadequate doses of levothyroxine sodium and 18% to 24% receive excessive doses based on having serum TSH levels outside the reference range [0.4 to 4.0 mIU/L9].

Given these factors, it is vital that different levothyroxine sodium formulations be bioequivalent. To be bioequivalent, two formulations must be pharmaceutically equivalent and have equivalent therapeutic effects. To be pharmaceutically equivalent, formulations must have the same active ingredient, be of equal strength and have undergone similar, high-quality manufacturing processes. The issue of therapeutic equivalence between levothyroxine sodium formulations is directly related to the method of determining their bioequivalence. Between two formulations, bioequivalence is typically defined as the relative bioavailabilities of the active ingredient, which in turn, is related to the rate and extent of absorption of the active ingredient. To determine bioequivalence, the United States Food and Drug Administration (FDA) requires the completion of pharmacokinetic studies. Pharmacokinetic studies designed to assess bioequivalence are performed in healthy volunteers. Thyroxine is an endogenous hormone which is indistinguishable from exogenously administered levothyroxine sodium, both in its biochemical characteristics and physiologic effects. For pharmacokinetic studies measuring the bioavailability of levothyroxine sodium formulations, the FDA recommends a single, supratherapeutic dose (600 mcg) be administered to healthy subjects.2 In this manner, it is assumed that serum concentrations of levothyroxine sodium may be sufficiently above endogenous baseline thyroid hormone levels to achieve meaningful pharmacokinetic measurements. However, even at supratherapeutic doses, endogenous thyroid hormone levels continue to contribute significantly to pharmacokinetic measurements.

Levothyroxine sodium formulations are considered bioequivalent if the 90% Confidence Intervals for the ratios (test formulation/reference formulation) of the geometric mean area under the serum concentration-time curve (AUC) and the maximum serum concentration (Cmax) fall between 0.8 and 1.25.2

Assessment of Bioequivalence Methodology for Levothyroxine Sodium

Concern over the confounding effect of baseline endogenous thyroid hormone levels led investigators to question the methodology for assessing bioequivalence of levothyroxine sodium formulations. In an attempt to improve existing bioequivalence protocol, a study was designed to examine 3 different methods of mathematical correction intended to account for the endogenous contributions of thyroid hormone.10

Thirty-six volunteers (18 women and 18 men) between 19 and 50 years of age were enrolled into a single-dose, open-label, three-period, crossover pharmacokinetic study of levothyroxine sodium. Subjects were clinically and biochemically euthyroid and in good health based on the results of medical history, physical examination, 12-lead electrocardiogram, and routine laboratory tests. None of the women were pregnant or at risk for pregnancy; none were taking contraceptive agents or breastfeeding.

Patients were randomized to levothyroxine sodium (Synthroid) 600 mcg (reference dose), 450 mcg or 400 mcg for 3 study periods, with a washout interval of ≥44 days between periods. Doses were administered with 50 mcg tablets from the same manufactured lot of 50 mcg tablets.

Serum concentrations for total thyroxine and total triiodothyronine and TSH were measured the day before dosing, the day of dosing, and at predetermined time points up to 96 hours after dosing.

The pharmacokinetic parameters for thyroxine were determined using Cmax, time to Cmax (Tmax), and AUC from 0-48 hours (AUC48), 0-72 hours (AUC72), and 0-96 hours (AUC96). The values of these parameters were determined both with and without correction for endogenous thyroxine levels. Correction for endogenous thyroxine levels was applied with each of 3 methods:

Correction Method 1 - for each subject and period, the mean of 3 thyroxine values at -0.5, -0.25, and 0 hours before dosing was subtracted from each post-dose thyroxine concentration. This method assumes that the contribution of endogenous thyroxine is constant; that is, it does not account for diurnal variation in endogenously produced thyroxine or the fact that the amount of endogenous thyroxine is constantly changing due to metabolism and excretion.

Correction Method 2 - for each subject and period, each thyroxine concentration after dosing was corrected for the hypothetical decay of endogenous thyroxine with a 7-day half-life, beginning with the level immediately before dosing. This method does not account for diurnal variation in endogenously produced thyroxine and further assumes that all endogenous thyroxine production ceases immediately following adminis-tration of the study dose.

Correction Method 3 - for each subject and period, each thyroxine concentration after dosing was adjusted by subtracting the thyroxine concentration measured at the analogous time-point on the day prior to administration of the study dose (Study Day 1) of each period. This method attempts to account for both diurnal variations in endogenously produced thyroxine and ongoing thyroxine metabolism and excretion. However, the method does not account for the interplay of exogenously administered levothyroxine sodium and endogenous thyroxine production.

Serum Thyroxine Levels with and without Correction for Endogenous Thyroid Hormone

Uncorrected Serum Thyroxine
The mean thyroxine serum concentration-time profiles after administration of each of the 400 mcg, 450 mcg, and 600 mcg doses of levothyroxine sodium are illustrated in Figure 1.

When no correction is made for endogenous thyroxine levels, both the 450 mcg and 400 mcg doses, in addition to being bioequivalent to each other, are bioequivalent to the 600 mcg dose as defined by FDA bioequivalence standards (0.8-1.25). Thus, the use of baseline uncorrected thyroxine Cmax, AUC48, AUC72, and AUC96 values resulted in a finding of bioequivalence of doses that were 25% and 33% lower than the reference dose (450 mcg and 400 mcg vs 600 mcg, respectively).

Correction Method 1
Throughout the 96 hours after dosing, the mean serum thyroxine concentrations after correction for endogenous baseline levels were higher for the 600 mcg dose than the 450 mcg and 400 mcg doses (Figure 2).

Neither the 450 mcg nor the 400 mcg dose was bioequivalent to the 600 mcg dose but were bioequivalent to each other. Thus, correction method 1 allowed distinction of doses that differed by 25% and 33% from the 600 mcg dose, but 450 mcg was found to be bioequivalent to 400 mcg.

Correction Method 2
Throughout the 96 hours after dosing, the mean serum thyroxine concentrations after correction for endogenous baseline levels were higher for the 600 mcg dose than the 450 mcg and 400 mcg doses (Figure 3).

This correction method also distinguished the 450 mcg and 400 mcg doses from the 600 mcg dose and the two doses were not bioequivalent to 600 mcg; however 450 mcg was found to be bioequivalent to 400 mcg.

Correction Method 3
Throughout the 96 hours after dosing, the mean serum thyroxine concentrations after correction for endogenous baseline levels were higher for the 600 mcg dose than the 450 mcg and 400 mcg doses (Figure 4).

As with correction methods 1 and 2, correction method 3 could not distinguish between the two doses that differed by 12.5% (450 mcg and 400 mcg) but when doses differed by 25% and 33% (450 mcg and 400 mcg vs 600 mcg, respectively), they would not be considered bioequivalent.

Clinical Implications

In this study, using guidance for bioequivalence in place at the time of the study, not accounting for endogenous thyroxine levels resulted in failing to identify differences between levothyroxine sodium products varying by as much as 25% to 33% in dosage strength. [Ed. In March 2003, the FDA announced guidance which incorporated a simple baseline correction method. This method is able to differentiate doses varying between 25% to 33%, but is unable to differentiate a dose difference of 12.5%.] Because levothyroxine sodium has a narrow therapeutic range, its safe and effective use requires careful titration and vigilant monitoring. Clinically, these findings have considerable implications.

This study demonstrates that unauthorized switching between levothyroxine sodium formulations, even those deemed bioequivalent by FDA standards, may result in sub- or supratherapeutic thyroid hormone levels. In patients stabilized at a particular dose, such changes could go undetected for long periods of time. Current guidelines recommend serum TSH monitoring every 6 to 12 months in stabilized patients.5 In patients undergoing titration, switching between levothyroxine sodium formulations that differ by 12.5% or more may complicate titration, as typical dose increments are 25 mcg to 50 mcg in otherwise healthy clinically hypothyroid adults and 12.5 mcg to 25 mcg in elderly hypothyroid patients with existing cardiac disease.11

Subtherapeutic levothyroxine sodium doses result in potential cardiovascular problems such as hypercholesterolemia,12 increased fibrinolytic activity,13 systolic and diastolic dysfunction,14,15 atherosclerosis, and myocardial infarction.16,17 Supratherapeutic doses increase the likelihood of arrhythmias, ventricular dysfunction, reduced exercise performance, and possible cardiovascular death.7,17,18 In the elderly, iatrogenic hyperthyroidism has been associated with the development of osteoporosis.19


Levothyroxine sodium is a narrow therapeutic range agent that requires careful dosage selection and titration. Recent evidence suggests that bioequivalence standards may not distinguish between significantly different levothyroxine sodium doses, likely because the standards do not fully account for the complex contributions of endogenous thyroxine. Corrections designed to account for the contributions of endogenous thyroxine may be inadequate to detect clinically significant dose differences. Therefore, further study is warranted to come to a definitive conclusion for the most practical approach to correct for endogenous thyroxine. For these reasons, clinicians must carefully choose and monitor replacement therapy with levothyroxine sodium. Current guidelines emphasize the importance of maintaining patients on the same levothyroxine sodium formulation throughout treatment. This study underscores why this is an important guidance for the clinician to follow.


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15. Brenta G, Mutti LA, Schnitman M, Fretes O, Perrone A, Matute ML. Assessment of left ventricular diastolic function by radionuclide ventriculography at rest and exercise in subclinical hypothyroidism, and its response to L-thyroxine therapy. Am J Cardiol. 2003;91:1327-1330.
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Stephen H. Schneider, MD
No significant relationships to disclose.

This report contains no information on commercial products that are unlabeled for use or investigational uses of products not yet approved.

The opinions expressed in this publication are those of the participating faculty and do not necessarily reflect the opinions or the recommendations of their affiliated institutions: University of Medicine & Dentistry of New Jersey; MMC, Inc.; or any other persons. Any procedures, medications, or other courses of diagnosis or treatment discussed or suggested in this publication should not be used by clinicians without evaluation of their patients' conditions, assessment of possible contraindications or dangers in use, review of any applicable manufacturer's product information, and comparison with the recommendation of other authorities. This Hypothyroidism Express Report does not include discussion of treatment and indications outside of current approved labeling. This Hypothyroidism Express Report was made possible through an educational grant from Abbott Laboratories.

© 2004 Millennium Medical Communications, Inc. and UMDNJ-Center for Continuing and Outreach Education