Appendix A

Abbott Laboratories
Study M02-417 Synopsis and Discussion

Title of Study

Evaluating the Impact of Correcting for Endogenous T₄ Baseline on the Bioequivalence of Levothyroxine Sodium Formulations in Healthy Volunteers

Objective

The objective of this study was to evaluate the impact of various methods for correcting for endogenous T₄ baseline on the bioequivalence of levothyroxine sodium formulations in healthy volunteers.

Methodology

This Phase 1, single-dose, open-label, study was conducted according to a three-period, randomized crossover design in healthy volunteers. The total dose given was 600 µg levothyroxine sodium for Regimen A, 450 µg levothyroxine sodium for Regimen B and 400 µg levothyroxine sodium for Regimen C. Subjects received one of six sequences of Regimen A (twelve 50 µg Synthroid^® tablets), Regimen B (nine 50 µg Synthroid^® tablets) or Regimen C (eight 50 µg Synthroid^® tablets) under fasting conditions at approximately 0830 on Study Day 1 of each period. A washout interval of at least 44 days separated the doses of the three study periods.

Blood samples (sufficient to provide approximately 2 mL serum) for total levothyroxine (T₄), total triiodothyronine (T₃) and thyroid stimulating hormone (TSH) assay were collected by venipuncture into 5 mL evacuated siliconized collection tubes as follows:

At approximately 0 hours and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12 and 18 hours after the 0-hour collection on Study Day –1 in each study period.

At approximately –30 minutes, –15 minutes and at 0 hours prior to dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72 and 96 hours after dosing on Study Day 1 in each study period.

Serum concentrations of T₄ and T₃ were determined using validated radioimmunoassay (RIA) methods. The lower limit of quantification of T₄ was 1.00 µg/dL. The lower limit of quantification of T₃ was 0.25 ng/mL. Serum concentrations of TSH were determined using a validated IRMA assay; lower limit of quantification was 0.250 µIU/mL.

Subjects

Subjects were male and female volunteers between 19 and 50 years of age, inclusive. Subjects were judged to be euthyroid and in general good health based on the results of medical history, physical examination, vital signs, 12-lead electrocardiogram and laboratory tests. Females were postmenopausal, sterile, or if of childbearing potential, were not pregnant or breast-feeding and were practicing an acceptable method of birth control.

Thirty-six subjects (18 M, 18 F) participated in the study, with mean age of 32.9 years, mean weight of 74.5 kg and mean height of 172 cm. Three subjects received study drug in only one period and thus were not included in any of the pharmacokinetics analyses. Thirty-three subjects (16 M, 17 F) were included in the pharmacokinetic analyses, with mean age of 33.1 years, mean weight of 73.5 kg and mean height of 171 cm.

Pharmacokinetics and Statistical Methods

The pharmacokinetic parameters of total levothyroxine (T₄) were estimated using noncompartmental methods. These included: the maximum serum concentration (C_max) and time to C_max (T_max), the area under the serum concentration-time curve (AUC) from time 0 to 48 hours (AUC₄₈), time 0 to 72 hours (AUC₇₂) and time 0 to 96 hours (AUC₉₆). For T₄, values of these parameters (C_max, T_max, AUC₄₈, AUC₇₂ and AUC₉₆) were determined without correction for endogenous T₄ levels and after correcting all post-dose concentrations using each of following three methods:

Correction Method 1: The predose baseline value on the day of dosing was subtracted from each post-dose concentration. The pre-dose baseline value was calculated as the average of the three concentrations at –0.5, –0.25 and 0 hours prior to dosing in each period.

Correction Method 2: For each time of post-dose sampling, the observed concentration was corrected assuming that the endogenous T₄ baseline level at 0 hours declines according to a half-life of 7 days.

Correction Method 3: The T₄ concentration for each time of post-dose sampling was corrected by the concentration observed at the same time of day during the 24 hours preceding the dose.

For all three methods of correction, the corrected 0-hour concentration was assumed to be 0.

For uncorrected and corrected T₄ an analysis of variance (ANOVA) with fixed effects for sex, sequence, sex-by-sequence interaction, period, regimen and the interaction of sex with each of period and regimen, and with random effects for subjects nested within sex-by-sequence combination was performed for T_max, and the natural logarithms of C_max AUC₄₈, AUC₇₂ and AUC₉₆. A significance level of 0.05 was used for all tests.

The bioavailability of each of Regimen B (450 µg dose) and Regimen C (400 µg dose) relative to that of Regimen A (600 µg dose) for uncorrected and corrected T₄ was assessed by the two one-sided tests procedure¹ via 90% confidence intervals obtained from the analysis of the natural logarithms of AUC₄₈ and C_max. Bioequivalence was concluded if the 90% confidence intervals from the analyses of the natural logarithms of AUC₄₈ and C_max were within the 0.80 to 1.25 range. Likewise, the bioavailability of Regimen B (450 µg dose) relative to that of Regimen C (400 µg dose) was assessed. The same was done using each of AUC₇₂ and AUC₉₆ in place of AUC₄₈.

A repeated measures analysis was performed on the T₄ concentration data of Study
Day –1 for each period. To investigate the possibility of carryover effects, an ANOVA was performed on the logarithms of the Study Day –1 AUC₂₄.

Pharmacokinetic Results

Levothyroxine (T₄) Without Correcting for Endogenous T₄ Baseline Concentrations

The mean serum concentration-time plots for uncorrected T₄ after administration of levothyroxine sodium on Study Day 1 are presented in Figure 1. The mean T₄ serum concentrations-time profiles are fairly consistent after administration of the three regimens. Mean T₄ concentrations prior to dosing are approximately 7.5 µg/dL and increase to about 13 to 14 µg/dL at maximum before declining. The mean T₄ concentrations remain at approximately 9 µg/dL at 96 hours after administration of these large doses of levothyroxine sodium to the healthy volunteers.

Figure 1. Mean Levothyroxine (T₄) Concentration-Time Profiles on Study Day 1 Following Single Dose Administration of Levothyroxine Sodium – Uncorrected for Endogenous T₄ Baseline Concentrations

Mean ± standard deviation (SD) pharmacokinetic parameters of T₄ after administration of the three regimens without correcting for endogenous T₄ baseline concentrations are listed in Table 1.

Table 1. Mean ± SD Pharmacokinetic Parameters of Levothyroxine (T4) Without Correcting for Endogenous T4 Baseline Concentrations
		Regimens
Pharmacokinetic Parameters (units)		A: 600 µg Dose (N = 31)	B: 450 µg Dose (N = 33)	C: 400 µg Dose (N = 33)
Tmax	(h)	3.1 ± 2.4	3.2 ± 2.1	3.5 ± 3.3
Cmax	(µg/dL)	14.3 ± 2.14	13.2 ± 2.05^*	13.2 ± 2.45^*
AUC48	(µg•h/dL)	518 ± 71.8	493 ± 72.7^*	484 ± 73.6^*
AUC72	(µg•h/dL)	741 ± 102	712 ± 108^*	691 ± 102^*,+
AUC96	(µg•h/dL)	951 ± 133	919 ± 139	892 ± 133^*,+
* Statistically significantly different from Regimen A (ANOVA, p < 0.05). + Statistically significantly different from Regimen B (ANOVA, p < 0.05).

The bioequivalence/bioavailability results for uncorrected T₄ are listed in Table 2.

Table 2. Bioequivalence and Relative Bioavailability–Uncorrected Levothyroxine (T4)
Regimens				Relative Bioavailability
Test vs.	Pharmacokinetic	Central Value^*		Point	90% Confidence
Reference	Parameter	Test	Reference	Estimate^+	Interval
450 µg vs.600 µg	Cmax	13.0	14.0	0.928	0.890 – 0.968
	AUC48	481.7	504.8	0.954	0.927 – 0.982
	AUC72	694.9	721.9	0.963	0.936 – 0.990
	AUC96	896.2	925.6	0.968	0.941 – 0.996
400 µg vs. 600 µg	Cmax	12.9	14.0	0.921	0.883 – 0.960
	AUC48	469.6	504.8	0.930	0.904 – 0.958
	AUC72	670.4	721.9	0.929	0.903 – 0.955
	AUC96	865.7	925.6	0.935	0.909 – 0.962
450 µg vs. 400 µg	Cmax	13.0	12.9	1.007	0.967 – 1.050
	AUC48	481.7	469.6	1.026	0.997 – 1.055
	AUC72	694.9	670.4	1.037	1.009 – 1.065
	AUC96	896.2	865.7	1.035	1.007 – 1.064
* Antilogarithm of the least squares means for logarithms. + Antilogarithm of the difference (test minus reference) of the least squares means for logarithms.

Levothyroxine (T₄) After Correction for Endogenous T₄ Baseline Concentrations

The mean serum concentration-time plots for T₄, after correction for endogenous baseline levels of levothyroxine using each of the correction methods, are presented in Figure 2 for Correction Method 1, Figure 3 for Correction Method 2, and Figure 4 for Correction Method 3. The mean T₄ serum concentrations after correcting for endogenous baseline levels by any of the three methods of correction were higher after administration of Regimen A (600 µg dose) than after administration of Regimens B (450 µg dose) and C (400 µg dose) throughout the 96-hour sampling period. The mean baseline corrected T₄ concentrations for Regimens B (450 µg dose) and C (400 µg dose) were comparable throughout the 96-hour sampling period. The baseline corrected T₄ concentrations prior to dosing were assigned a value of zero for each of the three methods of correction. However, 96 hours after administration of these large doses of levothyroxine sodium to healthy volunteers the mean baseline corrected T₄ concentrations remain at approximately 1 to 2 µg/dL for Correction Methods 1 and 3 and approximately 3 to 4 µg/dL for Correction Method 2.

Figure 2. Mean Levothyroxine (T₄) Concentration-Time Profiles after Correction for Endogenous Baseline Levels of T₄ Using Correction Method 1

Figure 3. Mean Levothyroxine (T₄) Concentration-Time Profiles after Correction for Endogenous Baseline Levels of T₄ Using Correction Method 2

Figure 4. Mean Levothyroxine (T₄) Concentration-Time Profiles after Correction for Endogenous Baseline Levels of T₄ Using Correction Method 3

Mean ± SD pharmacokinetic parameters of T₄ after administration of the three regimens after correcting for endogenous T₄ baseline concentrations are listed in Table 3.

Table 3. Mean ± SD Pharmacokinetic Parameters of Levothyroxine (T4) after Correcting for Endogenous T4 Baseline Concentrations
		Regimens
Pharmacokinetic Parameters (units)		A: 600 µg Dose (N = 31)	B: 450 µg Dose (N = 33)	C: 400 µg Dose (N = 33)
Correction Method 1
Tmax	(h)	3.1 ± 2.4	3.2 ± 2.1	3.5 ± 3.3
Cmax	(µg/dL)	7.05 ± 1.66	5.54 ± 1.53^*	5.72 ± 1.44^*
AUC48	(µg•h/dL)	172 ± 40.4	126 ± 39.0^*	123 ± 45.4^*
AUC72	(µg•h/dL)	222 ± 56.0	161 ± 55.5^*	149 ± 68.6^*
AUC96	(µg•h/dL)	259 ± 72.5	184 ± 69.9^*	169 ± 92.5^*
Correction Method 2
Tmax	(h)	3.3 ± 2.8	5.8 ± 9.3	3.7 ± 3.5
Cmax	(µg/dL)	7.15 ± 1.64	5.68 ± 1.50^*	5.83 ± 1.45^*
AUC48	(µg•h/dL)	204 ± 40.9	160 ± 40.1^*	156 ± 43.4^*
AUC72	(µg•h/dL)	292 ± 56.9	235 ± 58.2^*	221 ± 62.7^*
AUC96	(µg•h/dL)	379 ± 74.0	312 ± 74.6^*	295 ± 82.2^*
Correction Method 3
Tmax	(h)	3.5 ± 3.1	3.6 ± 2.3	3.6 ± 4.0
Cmax	(µg/dL)	7.03 ± 1.64	5.85 ± 1.78^*	5.56 ± 1.69^*
AUC48	(µg•h/dL)	176 ± 36.9	131 ± 39.2^*	120 ± 28.4^*
AUC72	(µg•h/dL)	226 ± 49.4	166 ± 52.9^*	146 ± 45.4^*,+
AUC96	(µg•h/dL)	263 ± 64.8	189 ± 65.6^*	167 ± 67.2^*
* Statistically significantly different from Regimen A (ANOVA, p < 0.05). + Statistically significantly different from Regimen B (ANOVA, p < 0.05).

The bioequivalence/bioavailability results for T₄ using Correction Method 1, Correction Method 2, and Correction Method 3 are listed in Tables 4, 5, and 6, respectively.

Table 4. Bioequivalence and Relative Bioavailability for T4 (Correction Method 1)
Regimens				Relative Bioavailability
Test vs.	Pharmacokinetic	Central Value^*		Point	90% Confidence
Reference	Parameter	Test	Reference	Estimate^+	Interval
450 µg vs.600 µg	Cmax	5.4	6.9	0.783	0.727 – 0.844
	AUC48	119.7	167.3	0.715	0.658 – 0.778
	AUC72	151.4	215.7	0.702	0.636 – 0.774
	AUC96	170.2	250.2	0.680	0.602 – 0.768
400 µg vs. 600 µg	Cmax	5.6	6.9	0.803	0.745 – 0.865
	AUC48	118.9	167.3	0.711	0.653 – 0.773
	AUC72	144.9	215.7	0.672	0.609 – 0.741
	AUC96	165.1	250.2	0.660	0.584 – 0.746
450 µg vs. 400 µg	Cmax	5.4	5.6	0.975	0.906 – 1.049
	AUC48	119.7	118.9	1.007	0.926 – 1.094
	AUC72	151.4	144.9	1.044	0.948 – 1.150
	AUC96	170.2	165.1	1.031	0.914 – 1.163
* Antilogarithm of the least squares means for logarithms. + Antilogarithm of the difference (test minus reference) of the least squares means for logarithms.

Table 5. Bioequivalence and Relative Bioavailability for T4 (Correction Method 2)
Regimens						Relative Bioavailability
Test vs.	Pharmacokinetic	Central Value^*				Point		90% Confidence
Reference	Parameter	Test		Reference		Estimate^+		Interval
450 µg vs.600 µg	Cmax	5.6		7.0		0.793		0.739 – 0.850
	AUC48	154.5		199.1		0.776		0.721 – 0.835
	AUC72	227.5		284.9		0.799		0.729 – 0.875
	AUC96	301.6		369.5		0.816		0.743 – 0.897
400 µg vs. 600 µg	Cmax	5.7		7.0		0.807		0.753 – 0.866
	AUC48	148.4		199.1		0.745		0.693 – 0.802
	AUC72	207.9		284.9		0.730		0.666 – 0.800
	AUC96	277.3		369.5		0.750		0.683 – 0.824
450 µg vs. 400 µg	Cmax	5.6		5.7		0.982		0.916 – 1.051
	AUC48	154.5		148.4		1.041		0.969 – 1.119
	AUC72	227.5		207.9		1.094		1.001 – 1.197
	AUC96	301.6		277.3		1.088		0.992 – 1.192
* Antilogarithm of the least squares means for logarithms. + Antilogarithm of the difference (test minus reference) of the least squares means for logarithms.
Table 6. Bioequivalence and Relative Bioavailability for T4 (Correction Method 3)
Regimens					Relative Bioavailability
Test vs.	Pharmacokinetic	Central Value^*			Point		90% Confidence
Reference	Parameter	Test	Reference		Estimate^+		Interval
450 µg vs.600 µg	Cmax	5.7	6.9		0.820		0.757 – 0.888
	AUC48	125.1	172.9		0.723		0.672 – 0.779
	AUC72	158.7	222.0		0.715		0.645 – 0.792
	AUC96	177.7	256.6		0.693		0.631 – 0.760
400 µg vs. 600 µg	Cmax	5.3	6.9		0.775		0.715 – 0.839
	AUC48	115.4	172.9		0.667		0.620 – 0.718
	AUC72	135.9	222.0		0.612		0.553 – 0.678
	AUC96	164.0	256.6		0.639		0.582 – 0.702
450 µg vs. 400 µg	Cmax	5.7	5.3		1.058		0.979 – 1.145
	AUC48	125.1	115.4		1.084		1.008 – 1.165
	AUC72	158.9	135.9		1.168		1.057 – 1.291
	AUC96	177.7	164.0		1.084		0.989 – 1.188
* Antilogarithm of the least squares means for logarithms. + Antilogarithm of the difference (test minus reference) of the least squares means for logarithms.

Baseline Levothyroxine (T₄) Prior to Dosing (Study Day –1)

The mean serum concentration-time plots for baseline T₄ on Study Day –1 prior to dosing with levothyroxine sodium in each Period are presented in Figure 5. Analysis of the T₄ concentration data obtained during the 24 hours of Study Day –1 of each period confirmed that T₄ has a diurnal cycle with statistically significant differences across time. The diurnal variation in baseline T₄ concentrations prior to dosing are consistent with the observed diurnal variation in the serum concentrations of TSH (Figure 6).

Analysis of the 24-hour AUC for Study Day –1 revealed that the regimens (dose levels) had statistically significantly different carryover effects from one period to the next (first-order carryover) and from Period 1 to Period 3 (second-order carryover).

Figure 5. Mean Levothyroxine (T₄) Concentration-Time Profiles on Study Day –1 Prior to Dosing with Levothyroxine Sodium by Period

Thyroid-Stimulating Hormone (TSH)

The mean serum concentration-time plots for TSH for the 24 hours prior to and 96 hours after administration of levothyroxine sodium on Study Day 1 are presented in Figure 6. The serum concentrations of TSH appear to clearly show diurnal variation, prior to dosing. During the 24-hour period prior to dosing, the concentrations of TSH decline during the morning hours until reaching the lowest levels at approximately 1200 before starting to increase to maximum values at 0200 the next morning, i.e., the morning of Study Day 1 (18 hour sample on Study Day –1).

Administration of any of the three large doses of levothyroxine sodium substantially, but not completely, suppressed the TSH serum concentrations throughout the 24-hour period after dosing on Study Day 1. TSH serum concentrations continued to be suppressed throughout the 96-hour sampling period after dosing; the concentrations did not return to baseline values even after 96 hours. The rank order of suppression of the TSH serum concentrations was consistent with the rank order of the size of levothyroxine sodium dose administered in each of the three regimens with the greatest suppression of TSH serum concentrations associated with administration of the largest dose (Regimen A, 600 µg).

Figure 6. Mean TSH Concentration-Time Profiles for the 24 Hours Prior to (Study Day –1) and for the 96 Hours after Administration of Levothyroxine Sodium on Study Day 1

Triiodothyronine (T₃) Concentrations

The mean T₃ concentration for the 24-hour period prior to dosing and throughout the 96‑hour period after dosing were in the very narrow range of 1.1 to 1.3 ng/mL after administration of the large doses of levothyroxine sodium to healthy volunteers.

Discussion

Determination of the bioavailability of levothyroxine sodium products in healthy volunteers presents significant challenging issues. Levothyroxine is naturally present in the blood, with total endogenous baseline T₄ levels ranging from 4 to 14 µg/dL. Thus, to compare the bioavailabilities of levothyroxine sodium formulations after a single dose in healthy volunteers, FDA Guidance² recommends administration of 600 µg, several times the normal clinical dose, to raise the levels of the drug significantly above baseline and to hopefully reduce the influence of endogenous levels. However, results from several bioavailability studies and a stochastic simulation study with levothyroxine products suggested that, given very reasonable assumptions about endogenous levothyroxine behavior in healthy subjects, the use of baseline uncorrected C_max and AUC₄₈ values would result in a high probability of declaring two products bioequivalent when they actually differ by as much as 35%.³

The current study was designed to evaluate how much two formulations could differ and still pass the bioequivalence criteria specified in the current guidance when not correcting for endogenous T₄ baseline levels. The results from this study clearly indicate that the use of baseline uncorrected C_max, AUC₄₈, AUC₇₂ and AUC₉₆ values would result in declaring two products bioequivalent when they actually differ by as much as 25% to 33% (450 µg and 400 µg versus 600 µg). Utilizing the criteria specified in FDA Guidance,² both the 450 µg dose (Regimen B) and the 400 µg dose (Regimen C) would be declared bioequivalent to the 600 µg dose (Regimen A) because the 90% confidence intervals for evaluating bioequivalence obtained without correcting for endogenous T₄ baseline levels were contained within the 0.80 to 1.25 range. Furthermore, the 450 µg dose would be declared bioequivalent to the 400 µg dose because the 90% confidence intervals for evaluating bioequivalence without correcting for endogenous T₄ baseline levels were contained within the 0.80 to 1.25 range. Considering the margin by which the conditions for declaring bioequivalence were passed in this study, products that differ by more than 33% would have a good chance of being declared bioequivalent on the basis of uncorrected data. The results of this study clearly demonstrate the significant limitations and problems with the current methodology and criteria for assessing the bioequivalence of levothyroxine sodium products in healthy volunteers without correcting for endogenous T₄ baseline levels.

Several mathematical and statistical methods can be used to correct for the contribution of T₄ baseline levels, based on different biologic assumptions about the behavior of endogenous T₄ following administration of exogenous levothyroxine. When a single dose of exogenous levothyroxine sodium is given to healthy subjects, one could assume that endogenous levothyroxine levels remain constant if there is no suppression of endogenous production (Correction Method 1). If production were completely suppressed, via feedback through the hypothalamic-pituitary axis, the endogenous levothyroxine would decline at an average rate defined by its half-life, which is approximately 7 days (Correction Method 2). Thus, a constant baseline of endogenous levothyroxine (Correction Method 1) versus a baseline that decays exponentially with a 7-day half-life (Correction Method 2) defines the limits for endogenous levothyroxine following a dose of exogenous levothyroxine sodium. This assumes that no other components of the thyroid system would impact the turnover of T₄ and T₃. The third method of baseline correction (Correction Method 3) employed in this study corrected the T₄ concentration for each time of post-dose sampling by the baseline T₄ concentration observed at the same time of day during the 24 hours preceding the dose, i.e., on Study Day –1.

One of the objectives of the current study was to better understand the impact of three different methods of correction for endogenous T₄ baseline on the bioequivalence evaluation of levothyroxine sodium formulations in healthy volunteers. In contrast to the results with uncorrected data, for all three correction methods for endogenous T₄ baseline, neither the 450 µg dose nor the 400 µg dose would be declared bioequivalent to the 600 µg dose. However, as with the uncorrected data, the 450 µg dose would continue to be declared bioequivalent to the 400 µg dose after correcting for endogenous T₄ baseline levels using any of the three correction methods because the 90% confidence intervals for evaluating bioequivalence after correcting for endogenous T₄ baseline continue to be contained within the 0.80 to 1.25 range. The 50µg difference between the 450 µg dose and the 400 µg dose represents a 12.5% difference.

Correction Method 1 relies on the assumption that there is no suppression of endogenous production when a single large dose of exogenous levothyroxine sodium is given to healthy subjects, thus assuming a constant baseline of endogenous levothyroxine. This assumption is clearly not true since TSH levels after dosing with levothyroxine sodium in the study were definitely suppressed, though not completely. Thus, it is very unlikely that endogenous T₄ production would be constant after administration of large doses of levothyroxine sodium to healthy volunteers. This method of correction has also several undesirable characteristics. The method will sometimes produce a negative value for AUC as was observed with one of the subjects in this study. Furthermore, the method relies completely upon the results from only three samples obtained during an interval of only 30 minutes just prior to dosing. Just from a consideration of randomness alone, the influence of the average of these three concentrations could be significant. More troubling than the small number of observations is the brief time span from which they are taken. It is known that there is a circadian effect on hormone levels, and the Day –1 data from this study clearly confirmed the presence of the circadian effect. Therefore, unless a subject's expected T₄ levels during the 30 minute time frame just prior to dosing happens also to be the expected average for a 24-hour cycle, the corrected AUC by this method is in error.

Correction Method 2 depends upon the assumption that endogenous production of levothyroxine is completely suppressed when a single large dose of exogenous levothyroxine sodium is given to healthy subjects. Therefore, already available endogenous levothyroxine will decline at rate defined by its half-life, which is assumed to be 7 days. This method also has several undesirable characteristics. Method 2 gives a reasonable correction only if production of endogenous T₄ abruptly and completely stops when study drug is administered and does not resume during the sampling period. Even if this unlikely assumption is true, the correction will be in error for a given subject, with the size of the error depending on how much the given subject's elimination half-life differs from 7 days. The half-life of levothyroxine is not very well documented in healthy volunteers and the 7-day half-life is an approximation based on data from isotope studies with levothyroxine. As previously noted, TSH levels after dosing with levothyroxine sodium were definitely suppressed, but not completely. Thus, it seems very unlikely that endogenous T₄ production would be reduced to zero, with an accompanying 7‑day half-life. The use of a single value for levothyroxine half-life for all healthy subjects (regardless of gender, race, and age) at all times is clearly a significant oversimplification. However, estimation of a levothyroxine half-life for each subject in each period is not possible using the currently recommended design in healthy volunteers. Moreover, as with Method 1, Method 2 relies heavily on the average of three concentrations taken immediately before dosing. In particular, for the case in which a subject randomly has a pre-dose average considerably higher than typical for that subject, the corrected AUC is more likely to be negative.

The third method of baseline correction (Method 3) employed in this study corrected the T₄ concentration at each time of post-dose sampling by the corresponding baseline T₄ concentration observed at the same time of day during the 24-hour period preceding the dose, i.e., on Study Day –1. This method provides some advantages in comparison to Methods 1 and 2. The obvious advantages for this method are a) it does not rely on just three samples collected over a very short time period prior to dosing for the correction, and b) the post-dose T₄ concentration is adjusted based on the actual baseline T₄ concentration at the same clock time of the day before dosing in the same subject in the same period, and thus, this method takes into account the diurnal variation in the baseline T₄ concentration throughout the day in each subject, which is ignored by Methods 1 and 2.

In contrast to Method 2, for Method 3, endogenous T₄ production is not assumed to abruptly stop following study drug administration and a constant value for the elimination half-life across subjects is not assumed. However, similar to Method 1, Method 3 relies on the assumption that there is no suppression of endogenous production when a single dose of exogenous levothyroxine sodium is given to healthy volunteers. Furthermore, Method 3 requires the assumption that the circadian pattern in the endogenous T₄ production does not change when a single large dose of exogenous levothyroxine is administered to healthy subjects.

The impact of administration of large doses of levothyroxine sodium (e.g., 600 µg) on the endogenous production of T₄ is not known. However, the TSH levels are clearly, but not completely, suppressed after administration of the large doses of levothyroxine sodium to the healthy volunteers in this study. The large exogenous dose may also affect the clearance of total T₄ via numerous feedback mechanisms. The TSH serum concentration-time data provide clear evidence of the limitations for each of the three methods of correction utilized in this study. Method 2 assumes that endogenous T₄ production is abruptly and completely stopped after study drug administration while Methods 1 and 3 assume that there is no suppression of endogenous production when a single dose of exogenous levothyroxine sodium is given to healthy volunteers.

The FDA Guidance² recommended a minimum 35-day washout period between the doses of levothyroxine sodium to minimize carryover. The 24-hour profiles of the baseline T₄ serum concentrations on the day before dosing were clearly not the same for the three study periods even though the washout periods between the doses of levothyroxine sodium in this study were 44 days between Periods 1 and 2 and 53 days between Periods 2 and 3. The Day –1 baseline T₄ data from this study provide convincing evidence that there are carryover effects from the successive study doses, even from the Period 1 dose to the Period 3 dose, and that the carryover effects of the dose levels differ. Carryover effect from the 600 µg dose resulted in higher T₄ levels than carryover effects of the two lower doses. Exploratory analyses of post-dose uncorrected C_max and AUC give additional strong evidence of these carryover effects. Also, such unequal carryover effects are present for C_max with all three methods of correction. Another component of the period effect may be the presence of seasonal and annual variations in hypothalamic-pituitary-thyroid hormone concentrations in humans. Significant seasonal and annual rhythms in serum TSH and T₃ levels have been reported in the literature.⁴ However, the amplitude of the circannual rhythm is probably not as large as that of the daily circadian variation.⁴ Therefore, the results from our studies suggest that a much longer washout period between dosing would be required to truly reduce the impact of carryover between dosing periods.

The results of this study strongly suggest that obtaining additional blood samples on Study Day –1 provided data that improved the method of correction for endogenous levels of T₄, accounting for the possibility of a circadian pattern. Additional samples during the afternoon and night hours on the day before dosing and on the days after dosing may provide further benefits to this method of correcting for the endogenous baseline.

It is widely recognized that dose initiation and titration need to be done in susceptible groups with the 12.5 µg dosage strength. In the package insert of levothyroxine sodium products,⁵ it states under 'Dosage and Administration – Specific Patient Populations' "the recommended starting dose of levothyroxine sodium in elderly patients with cardiac disease is 12.5 – 25 µg/day, with gradual dose increments at 4 to 6 week intervals. The levothyroxine sodium dose is generally adjusted in 12.5 to 25 µg increments until the patient with primary hypothyroidism is clinically euthyroid and the serum TSH has normalized." NDA approved levothyroxine sodium tablets are available in strengths that differ from their nearest doses by 12 to 13 µg/tablet: that is 75, 88, 100, 112, 125, 137 and 150 µg tablet strengths. The 88 and 112 µg strengths are 12% less or greater, respectively, than the 100 µg strength.

Even though the three methods of correction for endogenous T₄ baseline improve the ability to distinguish between products that are truly different in dose by 25% to 33%, none of the three correction methods were able to distinguish between two products that differ by 12.5%. As stated earlier and similar to the findings with the uncorrected data, the 450 µg dose would continue to be declared bioequivalent to the 400 µg dose after correcting for endogenous T₄ baseline using any of the three correction methods. Narrowing the 90% confidence intervals for evaluating bioequivalence after correcting for endogenous T₄ baseline from the standard range of 0.80 to 1.25 would reduce the chance that two products that differ by 12.5% would be declared bioequivalent.

The potential for conducting bioequivalence trials in athyreotic subjects, a model that minimizes confounding effects from endogenous T₄ due to the absence of residual endogenous hormone, must also be considered. A study in athyreotic subjects would presumably be a multiple-dose study and long enough to properly address the issue of carryover effect. Such a study in athyreotic subjects would utilize therapeutic doses of levothyroxine sodium and remove the need for a method of baseline correction.

Conclusions

This study illustrates some important flaws in the design and analysis of single-dose crossover studies in healthy volunteers to assess bioequivalence of levothyroxine sodium products, stemming from the significant and complex contribution of endogenous T₄. First, the results indicate that the use of baseline uncorrected T₄ C_max, AUC₄₈, AUC₇₂ and AUC₉₆ values would result in declaring two products bioequivalent when they actually differ by as much as 25% to 33% (450 µg and 400 µg versus 600 µg). The 450 µg dose and the 400 µg dose would both be declared bioequivalent to the 600 µg dose because the 90% confidence intervals for evaluating bioequivalence without correction for endogenous T₄ baseline were contained within the 0.80 to 1.25 range. Considering the margin by which the conditions for declaring bioequivalence were passed in this study, products that differ by even more than 33% would also have a high likelihood of being declared bioequivalent.

Second, the results from this study indicate that the use of baseline corrected C_max, AUC₄₈, AUC₇₂ and AUC₉₆ values would reduce the likelihood that two products would be declared bioequivalent when they actually differ by 25% to 33%. After correcting for endogenous T₄ levels using each of the three correction methods employed in this study, neither the 450 µg dose nor the 400 µg dose would be declared bioequivalent to the 600 µg dose because the 90% confidence intervals for evaluating bioequivalence were not contained within the 0.80 to 1.25 range for C_max, AUC₄₈, AUC₇₂ and AUC₉₆.

Third, the 450 µg dose would continue to be declared bioequivalent to the 400 µg dose utilizing the C_max, AUC₄₈, and AUC₉₆ values for the baseline corrected T₄ data by any of the three methods of correction. A 12.5% difference (400 µg versus 450 µg) in levothyroxine sodium products may have a clinically relevant adverse impact on patients. Thus, it is apparent that simple methods of correction for endogenous T₄ concentrations in healthy volunteers are inadequate since these concentrations not only fluctuate on a diurnal cycle but may also be differentially affected by products with different rates and extents of absorption. Additionally, there is evidence of significant carryover from one dosing period to subsequent periods even with washout periods up to 53 days.

The potential for conducting multiple-dose bioequivalence trials in athyreotic subjects, a model that minimizes confounding effects from endogenous T₄ due to the absence of residual endogenous hormone, must also be considered. Such a study in athyreotic subjects would utilize therapeutic doses of levothyroxine sodium and remove the need for a method of baseline correction.

Reference List for AppendixA

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2. Guidance for Industry: Levothyroxine sodium tablets – in vivo pharmacokinetic and bioavailability studies and in vitro dissolution testing. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, December 2000.

3. Riley S, Ludden TM, Simulation study to assess alternative bioavailability calculations, study designs and acceptance criteria for determining the bioequivalence of levothyroxine sodium tablets. GloboMax Technical Report, Project #KNP00500, April 2002.

4. Maes M, Mommen K, Hendrickx D, Peeters D, D'Hondt P, Ranjan R, et. al. Components of biological variation, including seasonality, in blood concentrations of TSH, TT3, FT4, PRL, cortisol and testosterone in healthy volunteers. Clin Endocrinol. 1997;46:587-598.

5. Synthroid (levothyroxine sodium tablets, USP). Physician Package Insert. Abbott Laboratories, Inc., North Chicago, IL. 03-5195-R1-Rev. July, 2002.