# 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 (Cmax) and time to Cmax (Tmax), 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 (Cmax, Tmax, 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:]{.underline}** 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:]{.underline}** 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:]{.underline}** 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 Tmax, and the natural logarithms of Cmax
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 Cmax. Bioequivalence was concluded
if the 90% confidence intervals from the analyses of the natural
logarithms of AUC48 and Cmax 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.

  -----------------------------------------------------------------------
  ![](media/image1.wmf)

  -----------------------------------------------------------------------

> 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.

+-------+--------+-----------------+-----------------+----------------+
| Tab   |        |                 |                 |                |
| le 1. |        |                 |                 |                |
| Mean  |        |                 |                 |                |
| ± SD  |        |                 |                 |                |
| Pharm |        |                 |                 |                |
| acoki |        |                 |                 |                |
| netic |        |                 |                 |                |
| Param |        |                 |                 |                |
| eters |        |                 |                 |                |
| of    |        |                 |                 |                |
| Lev   |        |                 |                 |                |
| othyr |        |                 |                 |                |
| oxine |        |                 |                 |                |
| (T4)  |        |                 |                 |                |
| Wi    |        |                 |                 |                |
| thout |        |                 |                 |                |
| Corre |        |                 |                 |                |
| cting |        |                 |                 |                |
| for   |        |                 |                 |                |
| Endog |        |                 |                 |                |
| enous |        |                 |                 |                |
| T4    |        |                 |                 |                |
| Bas   |        |                 |                 |                |
| eline |        |                 |                 |                |
| Conc  |        |                 |                 |                |
| entra |        |                 |                 |                |
| tions |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
|       |        | Regimens        |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Pharm |        | A: 600 µg Dose\ | B: 450 µg Dose\ | C: 400 µg      |
| acoki |        | (N = 31)        | (N = 33)        | Dose\          |
| netic |        |                 |                 | (N = 33)       |
| Param |        |                 |                 |                |
| eters |        |                 |                 |                |
| (u    |        |                 |                 |                |
| nits) |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Tmax  | \(h\)  | 3.1 ± 2.4       | 3.2 ± 2.1       | 3.5 ± 3.3      |
+-------+--------+-----------------+-----------------+----------------+
| Cmax  | (      | 14.3 ± 2.14     | 13.2 ± 2.05\*   | 13.2 ± 2.45\*  |
|       | µg/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC48 | (µg    | 518 ± 71.8      | 493 ± 72.7\*    | 484 ± 73.6\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC72 | (µg    | 741 ± 102       | 712 ± 108\*     | 691 ± 102\*,+  |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC96 | (µg    | 951 ± 133       | 919 ± 139       | 892 ± 133\*,+  |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| \*    |        |                 |                 |                |
| Sta   |        |                 |                 |                |
| tisti |        |                 |                 |                |
| cally |        |                 |                 |                |
| sig   |        |                 |                 |                |
| nific |        |                 |                 |                |
| antly |        |                 |                 |                |
| diff  |        |                 |                 |                |
| erent |        |                 |                 |                |
| from  |        |                 |                 |                |
| Re    |        |                 |                 |                |
| gimen |        |                 |                 |                |
| A     |        |                 |                 |                |
| (A    |        |                 |                 |                |
| NOVA, |        |                 |                 |                |
| p \<  |        |                 |                 |                |
| 0     |        |                 |                 |                |
| .05). |        |                 |                 |                |
|       |        |                 |                 |                |
| \+    |        |                 |                 |                |
| Sta   |        |                 |                 |                |
| tisti |        |                 |                 |                |
| cally |        |                 |                 |                |
| sig   |        |                 |                 |                |
| nific |        |                 |                 |                |
| antly |        |                 |                 |                |
| diff  |        |                 |                 |                |
| erent |        |                 |                 |                |
| from  |        |                 |                 |                |
| Re    |        |                 |                 |                |
| gimen |        |                 |                 |                |
| B     |        |                 |                 |                |
| (A    |        |                 |                 |                |
| NOVA, |        |                 |                 |                |
| p \<  |        |                 |                 |                |
| 0     |        |                 |                 |                |
| .05). |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+

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

+-------------+-------------+----+--------+---------+----------------+
| Table 2.    |             |    |        |         |                |
| Bio         |             |    |        |         |                |
| equivalence |             |    |        |         |                |
| and         |             |    |        |         |                |
| Relative    |             |    |        |         |                |
| Bioava      |             |    |        |         |                |
| ilability-- |             |    |        |         |                |
| Uncorrected |             |    |        |         |                |
| Le          |             |    |        |         |                |
| vothyroxine |             |    |        |         |                |
| (T4)        |             |    |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
| Regimens    |             |    |        | R       |                |
|             |             |    |        | elative |                |
|             |             |    |        | B       |                |
|             |             |    |        | ioavail |                |
|             |             |    |        | ability |                |
+-------------+-------------+----+--------+---------+----------------+
| Test *vs.*  | Phar        | C  |        | Point   | 90% Confidence |
|             | macokinetic | en |        |         |                |
|             |             | tr |        |         |                |
|             |             | al |        |         |                |
|             |             | V  |        |         |                |
|             |             | al |        |         |                |
|             |             | ue |        |         |                |
|             |             | \* |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
| Reference   | Parameter   | Te | Ref    | Es      | Interval       |
|             |             | st | erence | timate+ |                |
+-------------+-------------+----+--------+---------+----------------+
| 450 µg      | Cmax        | 13 | 14.0   | 0.928   | 0.890 -- 0.968 |
| *vs.*600 µg |             | .0 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC48       | 4  | 504.8  | 0.954   | 0.927 -- 0.982 |
|             |             | 81 |        |         |                |
|             |             | .7 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC72       | 6  | 721.9  | 0.963   | 0.936 -- 0.990 |
|             |             | 94 |        |         |                |
|             |             | .9 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC96       | 8  | 925.6  | 0.968   | 0.941 -- 0.996 |
|             |             | 96 |        |         |                |
|             |             | .2 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
| 400 µg      | Cmax        | 12 | 14.0   | 0.921   | 0.883 -- 0.960 |
| *vs.* 600   |             | .9 |        |         |                |
| µg          |             |    |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC48       | 4  | 504.8  | 0.930   | 0.904 -- 0.958 |
|             |             | 69 |        |         |                |
|             |             | .6 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC72       | 6  | 721.9  | 0.929   | 0.903 -- 0.955 |
|             |             | 70 |        |         |                |
|             |             | .4 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC96       | 8  | 925.6  | 0.935   | 0.909 -- 0.962 |
|             |             | 65 |        |         |                |
|             |             | .7 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
| 450 µg      | Cmax        | 13 | 12.9   | 1.007   | 0.967 -- 1.050 |
| *vs.* 400   |             | .0 |        |         |                |
| µg          |             |    |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC48       | 4  | 469.6  | 1.026   | 0.997 -- 1.055 |
|             |             | 81 |        |         |                |
|             |             | .7 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC72       | 6  | 670.4  | 1.037   | 1.009 -- 1.065 |
|             |             | 94 |        |         |                |
|             |             | .9 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
|             | AUC96       | 8  | 865.7  | 1.035   | 1.007 -- 1.064 |
|             |             | 96 |        |         |                |
|             |             | .2 |        |         |                |
+-------------+-------------+----+--------+---------+----------------+
| \*          |             |    |        |         |                |
| An          |             |    |        |         |                |
| tilogarithm |             |    |        |         |                |
| of the      |             |    |        |         |                |
| least       |             |    |        |         |                |
| squares     |             |    |        |         |                |
| means for   |             |    |        |         |                |
| logarithms. |             |    |        |         |                |
|             |             |    |        |         |                |
| \+          |             |    |        |         |                |
| An          |             |    |        |         |                |
| tilogarithm |             |    |        |         |                |
| 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.

  -----------------------------------------------------------------------
  ![](media/image2.wmf)

  -----------------------------------------------------------------------

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

  -----------------------------------------------------------------------
  ![](media/image3.wmf)

  -----------------------------------------------------------------------

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

  -----------------------------------------------------------------------
  ![](media/image4.wmf)

  -----------------------------------------------------------------------

> 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.

+-------+--------+-----------------+-----------------+----------------+
| Tab   |        |                 |                 |                |
| le 3. |        |                 |                 |                |
| Mean  |        |                 |                 |                |
| ± SD  |        |                 |                 |                |
| Pharm |        |                 |                 |                |
| acoki |        |                 |                 |                |
| netic |        |                 |                 |                |
| Param |        |                 |                 |                |
| eters |        |                 |                 |                |
| of    |        |                 |                 |                |
| Lev   |        |                 |                 |                |
| othyr |        |                 |                 |                |
| oxine |        |                 |                 |                |
| (T4)  |        |                 |                 |                |
| after |        |                 |                 |                |
| Corre |        |                 |                 |                |
| cting |        |                 |                 |                |
| for   |        |                 |                 |                |
| Endog |        |                 |                 |                |
| enous |        |                 |                 |                |
| T4    |        |                 |                 |                |
| Bas   |        |                 |                 |                |
| eline |        |                 |                 |                |
| Conc  |        |                 |                 |                |
| entra |        |                 |                 |                |
| tions |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
|       |        | Regimens        |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Pharm |        | A: 600 µg Dose\ | B: 450 µg Dose\ | C: 400 µg      |
| acoki |        | (N = 31)        | (N = 33)        | Dose\          |
| netic |        |                 |                 | (N = 33)       |
| Param |        |                 |                 |                |
| eters |        |                 |                 |                |
| (u    |        |                 |                 |                |
| nits) |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Corre |        |                 |                 |                |
| ction |        |                 |                 |                |
| M     |        |                 |                 |                |
| ethod |        |                 |                 |                |
| 1     |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Tmax  | \(h\)  | 3.1 ± 2.4       | 3.2 ± 2.1       | 3.5 ± 3.3      |
+-------+--------+-----------------+-----------------+----------------+
| Cmax  | (      | 7.05 ± 1.66     | 5.54 ± 1.53\*   | 5.72 ± 1.44\*  |
|       | µg/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC48 | (µg    | 172 ± 40.4      | 126 ± 39.0\*    | 123 ± 45.4\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC72 | (µg    | 222 ± 56.0      | 161 ± 55.5\*    | 149 ± 68.6\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC96 | (µg    | 259 ± 72.5      | 184 ± 69.9\*    | 169 ± 92.5\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Corre |        |                 |                 |                |
| ction |        |                 |                 |                |
| M     |        |                 |                 |                |
| ethod |        |                 |                 |                |
| 2     |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Tmax  | \(h\)  | 3.3 ± 2.8       | 5.8 ± 9.3       | 3.7 ± 3.5      |
+-------+--------+-----------------+-----------------+----------------+
| Cmax  | (      | 7.15 ± 1.64     | 5.68 ± 1.50\*   | 5.83 ± 1.45\*  |
|       | µg/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC48 | (µg    | 204 ± 40.9      | 160 ± 40.1\*    | 156 ± 43.4\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC72 | (µg    | 292 ± 56.9      | 235 ± 58.2\*    | 221 ± 62.7\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC96 | (µg    | 379 ± 74.0      | 312 ± 74.6\*    | 295 ± 82.2\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Corre |        |                 |                 |                |
| ction |        |                 |                 |                |
| M     |        |                 |                 |                |
| ethod |        |                 |                 |                |
| 3     |        |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| Tmax  | \(h\)  | 3.5 ± 3.1       | 3.6 ± 2.3       | 3.6 ± 4.0      |
+-------+--------+-----------------+-----------------+----------------+
| Cmax  | (      | 7.03 ± 1.64     | 5.85 ± 1.78\*   | 5.56 ± 1.69\*  |
|       | µg/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC48 | (µg    | 176 ± 36.9      | 131 ± 39.2\*    | 120 ± 28.4\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC72 | (µg    | 226 ± 49.4      | 166 ± 52.9\*    | 146 ± 45.4\*,+ |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| AUC96 | (µg    | 263 ± 64.8      | 189 ± 65.6\*    | 167 ± 67.2\*   |
|       | •h/dL) |                 |                 |                |
+-------+--------+-----------------+-----------------+----------------+
| \*    |        |                 |                 |                |
| Sta   |        |                 |                 |                |
| tisti |        |                 |                 |                |
| cally |        |                 |                 |                |
| sig   |        |                 |                 |                |
| nific |        |                 |                 |                |
| antly |        |                 |                 |                |
| diff  |        |                 |                 |                |
| erent |        |                 |                 |                |
| from  |        |                 |                 |                |
| Re    |        |                 |                 |                |
| gimen |        |                 |                 |                |
| A     |        |                 |                 |                |
| (A    |        |                 |                 |                |
| NOVA, |        |                 |                 |                |
| p \<  |        |                 |                 |                |
| 0     |        |                 |                 |                |
| .05). |        |                 |                 |                |
|       |        |                 |                 |                |
| \+    |        |                 |                 |                |
| Sta   |        |                 |                 |                |
| tisti |        |                 |                 |                |
| cally |        |                 |                 |                |
| sig   |        |                 |                 |                |
| nific |        |                 |                 |                |
| antly |        |                 |                 |                |
| diff  |        |                 |                 |                |
| erent |        |                 |                 |                |
| from  |        |                 |                 |                |
| Re    |        |                 |                 |                |
| gimen |        |                 |                 |                |
| B     |        |                 |                 |                |
| (A    |        |                 |                 |                |
| NOVA, |        |                 |                 |                |
| 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.    |             |    |   |       |         |                |
| Bio         |             |    |   |       |         |                |
| equivalence |             |    |   |       |         |                |
| and         |             |    |   |       |         |                |
| Relative    |             |    |   |       |         |                |
| Bioa        |             |    |   |       |         |                |
| vailability |             |    |   |       |         |                |
| for T4      |             |    |   |       |         |                |
| (Correction |             |    |   |       |         |                |
| Method 1)   |             |    |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
| Regimens    |             |    |   |       | R       |                |
|             |             |    |   |       | elative |                |
|             |             |    |   |       | B       |                |
|             |             |    |   |       | ioavail |                |
|             |             |    |   |       | ability |                |
+-------------+-------------+----+---+-------+---------+----------------+
| Test *vs.*  | Phar        | C  |   |       | Point   | 90% Confidence |
|             | macokinetic | en |   |       |         |                |
|             |             | tr |   |       |         |                |
|             |             | al |   |       |         |                |
|             |             | V  |   |       |         |                |
|             |             | al |   |       |         |                |
|             |             | ue |   |       |         |                |
|             |             | \* |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
| Reference   | Parameter   | Te |   | Refe  | Es      | Interval       |
|             |             | st |   | rence | timate+ |                |
+-------------+-------------+----+---+-------+---------+----------------+
| 450 µg      | Cmax        | 5  |   | 6.9   | 0.783   | 0.727 -- 0.844 |
| *vs.*600 µg |             | .4 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC48       | 1  |   | 167.3 | 0.715   | 0.658 -- 0.778 |
|             |             | 19 |   |       |         |                |
|             |             | .7 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC72       | 1  |   | 215.7 | 0.702   | 0.636 -- 0.774 |
|             |             | 51 |   |       |         |                |
|             |             | .4 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC96       | 1  |   | 250.2 | 0.680   | 0.602 -- 0.768 |
|             |             | 70 |   |       |         |                |
|             |             | .2 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
| 400 µg      | Cmax        | 5  |   | 6.9   | 0.803   | 0.745 -- 0.865 |
| *vs.* 600   |             | .6 |   |       |         |                |
| µg          |             |    |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC48       | 1  |   | 167.3 | 0.711   | 0.653 -- 0.773 |
|             |             | 18 |   |       |         |                |
|             |             | .9 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC72       | 1  |   | 215.7 | 0.672   | 0.609 -- 0.741 |
|             |             | 44 |   |       |         |                |
|             |             | .9 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC96       | 1  |   | 250.2 | 0.660   | 0.584 -- 0.746 |
|             |             | 65 |   |       |         |                |
|             |             | .1 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
| 450 µg      | Cmax        | 5  |   | 5.6   | 0.975   | 0.906 -- 1.049 |
| *vs.* 400   |             | .4 |   |       |         |                |
| µg          |             |    |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC48       | 1  |   | 118.9 | 1.007   | 0.926 -- 1.094 |
|             |             | 19 |   |       |         |                |
|             |             | .7 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC72       | 1  |   | 144.9 | 1.044   | 0.948 -- 1.150 |
|             |             | 51 |   |       |         |                |
|             |             | .4 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
|             | AUC96       | 1  |   | 165.1 | 1.031   | 0.914 -- 1.163 |
|             |             | 70 |   |       |         |                |
|             |             | .2 |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+
| \*          |             |    |   |       |         |                |
| An          |             |    |   |       |         |                |
| tilogarithm |             |    |   |       |         |                |
| of the      |             |    |   |       |         |                |
| least       |             |    |   |       |         |                |
| squares     |             |    |   |       |         |                |
| means for   |             |    |   |       |         |                |
| logarithms. |             |    |   |       |         |                |
|             |             |    |   |       |         |                |
| \+          |             |    |   |       |         |                |
| An          |             |    |   |       |         |                |
| tilogarithm |             |    |   |       |         |                |
| of the      |             |    |   |       |         |                |
| difference  |             |    |   |       |         |                |
| (test minus |             |    |   |       |         |                |
| reference)  |             |    |   |       |         |                |
| of the      |             |    |   |       |         |                |
| least       |             |    |   |       |         |                |
| squares     |             |    |   |       |         |                |
| means for   |             |    |   |       |         |                |
| logarithms. |             |    |   |       |         |                |
+-------------+-------------+----+---+-------+---------+----------------+

<table>
<colgroup>
<col style="width: 20%" />
<col style="width: 19%" />
<col style="width: 7%" />
<col style="width: 0%" />
<col style="width: 1%" />
<col style="width: 11%" />
<col style="width: 1%" />
<col style="width: 13%" />
<col style="width: 1%" />
<col style="width: 22%" />
<col style="width: 1%" />
</colgroup>
<tbody>
<tr class="odd">
<td colspan="11">Table 5. Bioequivalence and Relative Bioavailability
for T4 (Correction Method 2)</td>
</tr>
<tr class="even">
<td>Regimens</td>
<td></td>
<td></td>
<td colspan="4"></td>
<td colspan="4">Relative Bioavailability</td>
</tr>
<tr class="odd">
<td>Test <em>vs.</em></td>
<td>Pharmacokinetic</td>
<td colspan="5">Central Value*</td>
<td colspan="2">Point</td>
<td colspan="2">90% Confidence</td>
</tr>
<tr class="even">
<td>Reference</td>
<td>Parameter</td>
<td colspan="3">Test</td>
<td colspan="2">Reference</td>
<td colspan="2">Estimate+</td>
<td colspan="2">Interval</td>
</tr>
<tr class="odd">
<td>450 µg <em>vs.</em>600 µg</td>
<td>Cmax</td>
<td colspan="3">5.6</td>
<td colspan="2">7.0</td>
<td colspan="2">0.793</td>
<td colspan="2">0.739 – 0.850</td>
</tr>
<tr class="even">
<td></td>
<td>AUC48</td>
<td colspan="3">154.5</td>
<td colspan="2">199.1</td>
<td colspan="2">0.776</td>
<td colspan="2">0.721 – 0.835</td>
</tr>
<tr class="odd">
<td></td>
<td>AUC72</td>
<td colspan="3">227.5</td>
<td colspan="2">284.9</td>
<td colspan="2">0.799</td>
<td colspan="2">0.729 – 0.875</td>
</tr>
<tr class="even">
<td></td>
<td>AUC96</td>
<td colspan="3">301.6</td>
<td colspan="2">369.5</td>
<td colspan="2">0.816</td>
<td colspan="2">0.743 – 0.897</td>
</tr>
<tr class="odd">
<td>400 µg <em>vs.</em> 600 µg</td>
<td>Cmax</td>
<td colspan="3">5.7</td>
<td colspan="2">7.0</td>
<td colspan="2">0.807</td>
<td colspan="2">0.753 – 0.866</td>
</tr>
<tr class="even">
<td></td>
<td>AUC48</td>
<td colspan="3">148.4</td>
<td colspan="2">199.1</td>
<td colspan="2">0.745</td>
<td colspan="2">0.693 – 0.802</td>
</tr>
<tr class="odd">
<td></td>
<td>AUC72</td>
<td colspan="3">207.9</td>
<td colspan="2">284.9</td>
<td colspan="2">0.730</td>
<td colspan="2">0.666 – 0.800</td>
</tr>
<tr class="even">
<td></td>
<td>AUC96</td>
<td colspan="3">277.3</td>
<td colspan="2">369.5</td>
<td colspan="2">0.750</td>
<td colspan="2">0.683 – 0.824</td>
</tr>
<tr class="odd">
<td>450 µg <em>vs.</em> 400 µg</td>
<td>Cmax</td>
<td colspan="3">5.6</td>
<td colspan="2">5.7</td>
<td colspan="2">0.982</td>
<td colspan="2">0.916 – 1.051</td>
</tr>
<tr class="even">
<td></td>
<td>AUC48</td>
<td colspan="3">154.5</td>
<td colspan="2">148.4</td>
<td colspan="2">1.041</td>
<td colspan="2">0.969 – 1.119</td>
</tr>
<tr class="odd">
<td></td>
<td>AUC72</td>
<td colspan="3">227.5</td>
<td colspan="2">207.9</td>
<td colspan="2">1.094</td>
<td colspan="2">1.001 – 1.197</td>
</tr>
<tr class="even">
<td></td>
<td>AUC96</td>
<td colspan="3">301.6</td>
<td colspan="2">277.3</td>
<td colspan="2">1.088</td>
<td colspan="2">0.992 – 1.192</td>
</tr>
<tr class="odd">
<td colspan="11"><p>* Antilogarithm of the least squares means for
logarithms.</p>
<p>+ Antilogarithm of the difference (test minus reference) of the least
squares means for logarithms.</p></td>
</tr>
<tr class="even">
<td colspan="10">Table 6. Bioequivalence and Relative Bioavailability
for T4 (Correction Method 3)</td>
<td></td>
</tr>
<tr class="odd">
<td>Regimens</td>
<td></td>
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="4">Relative Bioavailability</td>
<td></td>
</tr>
<tr class="even">
<td>Test <em>vs.</em></td>
<td>Pharmacokinetic</td>
<td colspan="4">Central Value*</td>
<td colspan="2">Point</td>
<td colspan="2">90% Confidence</td>
<td></td>
</tr>
<tr class="odd">
<td>Reference</td>
<td>Parameter</td>
<td colspan="2">Test</td>
<td colspan="2">Reference</td>
<td colspan="2">Estimate+</td>
<td colspan="2">Interval</td>
<td></td>
</tr>
<tr class="even">
<td>450 µg <em>vs.</em>600 µg</td>
<td>Cmax</td>
<td colspan="2">5.7</td>
<td colspan="2">6.9</td>
<td colspan="2">0.820</td>
<td colspan="2">0.757 – 0.888</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC48</td>
<td colspan="2">125.1</td>
<td colspan="2">172.9</td>
<td colspan="2">0.723</td>
<td colspan="2">0.672 – 0.779</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>AUC72</td>
<td colspan="2">158.7</td>
<td colspan="2">222.0</td>
<td colspan="2">0.715</td>
<td colspan="2">0.645 – 0.792</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC96</td>
<td colspan="2">177.7</td>
<td colspan="2">256.6</td>
<td colspan="2">0.693</td>
<td colspan="2">0.631 – 0.760</td>
<td></td>
</tr>
<tr class="even">
<td>400 µg <em>vs.</em> 600 µg</td>
<td>Cmax</td>
<td colspan="2">5.3</td>
<td colspan="2">6.9</td>
<td colspan="2">0.775</td>
<td colspan="2">0.715 – 0.839</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC48</td>
<td colspan="2">115.4</td>
<td colspan="2">172.9</td>
<td colspan="2">0.667</td>
<td colspan="2">0.620 – 0.718</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>AUC72</td>
<td colspan="2">135.9</td>
<td colspan="2">222.0</td>
<td colspan="2">0.612</td>
<td colspan="2">0.553 – 0.678</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC96</td>
<td colspan="2">164.0</td>
<td colspan="2">256.6</td>
<td colspan="2">0.639</td>
<td colspan="2">0.582 – 0.702</td>
<td></td>
</tr>
<tr class="even">
<td>450 µg <em>vs.</em> 400 µg</td>
<td>Cmax</td>
<td colspan="2">5.7</td>
<td colspan="2">5.3</td>
<td colspan="2">1.058</td>
<td colspan="2">0.979 – 1.145</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC48</td>
<td colspan="2">125.1</td>
<td colspan="2">115.4</td>
<td colspan="2">1.084</td>
<td colspan="2">1.008 – 1.165</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>AUC72</td>
<td colspan="2">158.9</td>
<td colspan="2">135.9</td>
<td colspan="2">1.168</td>
<td colspan="2">1.057 – 1.291</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>AUC96</td>
<td colspan="2">177.7</td>
<td colspan="2">164.0</td>
<td colspan="2">1.084</td>
<td colspan="2">0.989 – 1.188</td>
<td></td>
</tr>
<tr class="even">
<td colspan="10"><p>* Antilogarithm of the least squares means for
logarithms.</p>
<p>+ Antilogarithm of the difference (test minus reference) of the least
squares means for logarithms.</p></td>
<td></td>
</tr>
</tbody>
</table>

### 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).

  -----------------------------------------------------------------------
  ![](media/image5.wmf)

  -----------------------------------------------------------------------

> 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).

  -----------------------------------------------------------------------
  ![](media/image6.wmf)

  -----------------------------------------------------------------------

> 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 Cmax 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 Cmax, 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 Cmax and
AUC give additional strong evidence of these carryover effects. Also,
such unequal carryover effects are present for Cmax 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]{.smallcaps} -- 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 Cmax, 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 Cmax, 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 Cmax, AUC48, AUC72 and AUC96.

Third, the 450 µg dose would continue to be declared bioequivalent to
the 400 µg dose utilizing the Cmax, 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

1.  Schuirman DJ. A comparison of the two one-sided tests procedure and
    the power approach for assessing the equivalence of average
    bioavailability. *J Pharmacokinetics Biopharm.* 1987;15:657-80.

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1.  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.

2.  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.

3.  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.

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