Comparative Pharmacokinetics of AFOLIA and US Gonal-f® RFF Redi-ject After Single Subcutaneous Application: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Comparative Pharmacokinetics of AFOLIA and US Gonal-f® RFF Redi-ject After Single Subcutaneous Application (NCT02459418), a randomized phase 1 study. The primary outcome is Baseline Corrected FSH Area Under the Serum Concentration-time Curve From Zero to the Last Quantifiable Measurement [AUC(0-last)].

2. Clinical Question

This is a pharmacokinetic comparison, not a trial of clinical fertility outcomes. Its primary endpoint captures the amount of baseline-corrected FSH in serum over the observed concentration-time profile, from predose through the last quantifiable measurement. The registry also lists secondary pharmacokinetic outcomes for FSH and 17ß-estradiol, including exposure, peak concentration, timing of the peak, and apparent terminal half-life.

The central inferential question is whether the estimated relative exposure and its uncertainty fall within the prespecified equivalence range described in the record. The posted sponsor analysis reports a geometric least-squares mean ratio of 164.96, with a 90% confidence interval from 137.82 to 197.45. These values are above the upper boundary of the stated 80%-125% range. This is a statistical reading of the reported pharmacokinetic comparison; it is not a conclusion about clinical benefit or treatment choice.

3. Trial Design

The registry describes a randomized, open-label, two-way crossover study in phase 1. It enrolled healthy volunteers and is marked completed. Fertility Biotech AG is listed as the sponsor. The recorded study start is 2015-05-07 and the primary completion date is 2016-05-19.

In a crossover design, each participant receives the study treatments in a sequence, so observations from the same participant can be compared across treatment periods. The record describes two sequences. Sequence A received a single subcutaneous injection of 225IU AFOLIA on study day 1, followed by a single subcutaneous injection of 225IU US Gonal-f® on study day 27. Sequence B received US Gonal-f® first and AFOLIA on study day 27. Random allocation determined the sequence; masking is recorded as none, so the study was open label.

The crossover structure is relevant to interpretation because a participant's responses under the treatments are not independent in the same way as responses from separate parallel groups. A suitable analysis can account for the paired, within-participant structure and for treatment sequence or period as specified in the statistical plan. The precise model and adjustment terms used for the sponsor's analysis are not reported in the registry record.

The registry reports enrollment of 42, while the posted primary endpoint summaries list 36 for AFOLIA and 33 for Gonal-f® RFF. Safety summaries use denominators of 36 for AFOLIA and 34 for Gonal-f® RFF. These are analysis-specific counts; they should not be treated as interchangeable. The reasons for differences among enrollment, pharmacokinetic, and safety counts are not reported in the registry record.

The schedule describes serial serum sampling after each dose, including predose and postdose time points through 192 hours. The study's objective was to compare pharmacokinetics after single applications, not to assess repeated treatment or clinical outcomes. Details such as any washout rationale, sample-size justification, and planned interim analyses are not reported in the registry record.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Before assignment: Screening was conducted at more than 1 visit to the clinical unit during study days -28 to -1 to allow completion of all assessments. Only subjects taking oral contraception for at least 3 months could enter the study. Subjects were required to stop taking their regular oral contraceptives so they could receive LupronDepot®.

Overall Study

Reasons for not completing this period:

Analysis population for the primary outcome, as defined in the registry: Analysis was performed on the Pharmacokinetic Analysis Set (PKAS) which includes all subjects who received active study drug and had at least 1 measured and valid concentration at a scheduled PK time point after administration of AFOLIA or Gonal-f® RFF. Only subjects with concentration data for determination of AUC(0-last) are included.

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

Population: All subjects randomized to receive study treatment.

6. Primary Endpoint

The registry lists the primary outcome as Baseline Corrected FSH Area Under the Serum Concentration-time Curve From Zero to the Last Quantifiable Measurement [AUC(0-last)], measured over From 0 (predose),0.5, 1, 3, 6, 9, 12, 16, 20, 21, 22, 23, 24, 25, 26, 27, 28, 48, 72, 96, 120, 144, 168 and 192 hours postdose..

The primary endpoint is baseline-corrected FSH area under the serum concentration-time curve from zero to the last quantifiable measurement, AUC(0-last), expressed in nanograms*hours/mL (ng*h/mL). An area under a concentration-time curve condenses a series of measurements into an estimate of overall exposure across the measured period. It is used in pharmacokinetic comparisons because it reflects the amount of circulating drug-related response over time, rather than relying on a single concentration.

For this endpoint, the record says baseline-corrected concentrations were obtained by subtracting the concentration measured immediately before dosing in that period from each postdose concentration. AUC(0-last) was estimated using noncompartmental methods and actual elapsed time from dosing. The scheduled sampling profile runs from predose through 192 hours after dosing. The endpoint therefore depends on the concentration measurements and on the observed time of the last quantifiable measurement; it is not an estimate of exposure beyond the last quantifiable observation.

The registry reports geometric means for the endpoint. Geometric summaries are often used for pharmacokinetic measures that are positive and may be skewed, because analysis on a logarithmic scale can make relative comparisons more appropriate. The stated objective is equivalence within 80%-125% of the reference product for AUC. The endpoint is useful for that objective because the comparison concerns relative exposure, but the endpoint alone does not establish equivalence: the estimated treatment ratio and its confidence interval must be interpreted against the stated margin.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

The registry identifies the primary measure as a geometric mean and gives its dispersion as the full range. It also reports a geometric least-squares mean ratio and a 90% confidence interval for the treatment comparison. These summaries answer related but distinct questions: the arm-level geometric means describe exposure within each treatment group, while the ratio expresses the estimated relative exposure in the sponsor's comparison. The confidence interval conveys uncertainty around that ratio under the analysis used.

What does AUC measure?

AUC is the area under the concentration-time curve. In general, it summarizes exposure across repeated measurements rather than describing only the highest concentration or a single sampling time. AUC(0-last) covers the period from the start of observation through the last quantifiable measurement. The registry describes baseline correction for FSH: the predose concentration for a period was subtracted from postdose concentrations. It also says AUC was estimated by noncompartmental methods using actual elapsed time from dosing. Those details matter because both the measured concentrations and the timing of samples contribute to the exposure estimate.

The primary summary is geometric. In general, geometric means are calculated on a logarithmic scale and returned to the original scale. This can be useful when pharmacokinetic measurements vary multiplicatively or have a right-skewed distribution. A geometric mean is not the same as an arithmetic average, and the full range is not a confidence interval. The ranges posted for AFOLIA and Gonal-f® RFF describe the observed spread of the endpoint values under each treatment.

How should the ratio and interval be read?

A ratio compares the estimated exposure under one treatment with the estimated exposure under the reference treatment. A ratio above the no-relative-difference point indicates a larger estimated value for the numerator treatment, given the comparison order. The registry labels the reported comparison as a geometric least-squares mean ratio and lists AFOLIA and Gonal-f® RFF as the comparison groups. Its estimate is 164.96, and the 90% confidence interval runs from 137.82 to 197.45.

In general, a confidence interval represents uncertainty in an estimate under a specified statistical model and its assumptions. It is not a range containing a fixed proportion of individual participants' values, and it is not a guarantee that the underlying value lies within the interval. For equivalence, the interval is judged against the acceptable bounds. The registry's stated range is 80%-125%; because the reported interval lies wholly above its upper limit, the stated equivalence objective is not met by this reported result.

Why does the crossover structure matter?

In a crossover trial, participants receive treatments in a sequence, making within-participant comparisons possible. In general, this can reduce the influence of stable differences between participants, because each participant can serve as their own comparison. It also calls for analysis that respects the repeated observations and the allocation to treatment sequence. Period effects, carryover, and the model used to estimate treatment effects can matter in crossover studies. The registry record describes randomized sequences and study days for dosing, but the full statistical model, any carryover assessment, and the handling of period or sequence effects are not reported in the registry record.

The posted values are analysis-set summaries, not a complete account of all randomized participants. The endpoint description specifies a Pharmacokinetic Analysis Set including people who received active study drug and had at least one measured and valid concentration at a scheduled pharmacokinetic time point. The final sentence of the population definition is incomplete in the registry record, so the full eligibility rule for the reported analysis cannot be established from that description.

What do the ranges and missing results add?

The full range reports the lowest and highest posted values within an arm; it does not quantify the precision of the treatment comparison. Wide ranges may reflect participant-to-participant variability, measurement features, or other influences, but the registry values alone do not identify the reasons for the observed spread. The record lists 8 outcome measures and 7 analyses, and it reports 6 secondary outcomes. It also says that values were missing or not estimable for a two-arm comparison for another endpoint. The identity of that endpoint and the reasons for non-estimability are not reported in the registry record.

General statistical caution is warranted when several outcomes are examined: repeated testing can increase the chance of chance findings unless the analysis plan addresses multiplicity. Whether and how multiplicity was handled is not reported in the registry record. Likewise, the registry does not report the missing-data methods, sample-size justification, or interim-analysis plan. These absences limit assessment of the prespecification and robustness of the analyses. They do not change the arithmetic meaning of the posted primary estimate, but they constrain how completely its analytic context can be evaluated.

10. Analyses Reported by the Sponsor

The registry reports the sponsor's primary comparison as a geometric least-squares mean ratio of 164.96, with a 90% confidence interval from 137.82 to 197.45. This is the analysis to use when describing the sponsor-reported equivalence result. Its interval lies above the upper boundary of the stated 80%-125% margin.

Arm-level geometric means are also posted: 18.96 for AFOLIA and 10.47 for Gonal-f® RFF. These are descriptive summaries and are not, by themselves, the sponsor's adjusted comparison. A simple unadjusted recalculation from arm summaries, when available, can differ from a model-based least-squares mean ratio because the methods may use different information and assumptions. Such a recalculation should not replace the sponsor's analysis.

The registry record does not identify a discrepancy between the sponsor-reported result and a recalculation. The full statistical model and its adjustment details are not reported in the registry record, so the exact relationship between any unadjusted calculation and the sponsor's model cannot be assessed from the record. No recalculated ratio or recalculated confidence interval is reported in the registry record. The posted sponsor estimate and interval are therefore the relevant figures for describing the stated primary analysis.

11. Multiplicity, Missing Data and Analysis Populations

The record lists 8 outcome measures, 7 analyses, and 6 secondary outcomes. The secondary outcomes include FSH exposure extrapolated to infinity, FSH time to maximum concentration, FSH apparent terminal half-life, and 17ß-estradiol exposure, peak concentration, and time to peak. The primary endpoint is baseline-corrected FSH AUC(0-last). These outcomes involve related but distinct pharmacokinetic characteristics.

When several outcomes are examined, statistical interpretation can be affected by multiplicity. In general, conducting multiple inferential tests without an appropriate strategy can increase the chance of at least one apparently notable result arising by chance. The registry record does not report whether a multiplicity adjustment or hierarchy was planned, or how secondary outcomes were to be interpreted. The posted primary result can be read against its stated equivalence margin, but the record does not provide enough information to evaluate family-wise error control across the full set of outcomes.

The registry says that the primary endpoint analysis used a Pharmacokinetic Analysis Set, defined as subjects who received active study drug and had at least one measured and valid concentration at a scheduled pharmacokinetic time point. The population description then ends with an incomplete additional eligibility phrase. Its full content is not reported in the registry record. The reason participants are absent from particular endpoint summaries is not reported in the registry record.

The arm counts differ by analysis: the primary pharmacokinetic summaries list 36 for AFOLIA and 33 for Gonal-f® RFF, while the safety summaries list 36 and 34. The registry does not explain these differences. It also notes that values were missing or not estimable for a two-arm comparison for another endpoint, without identifying the endpoint or giving the reason. The missing-data approach, sensitivity analyses, and handling of observations below quantification limits are not reported in the registry record. These gaps make it difficult to assess how missingness may have affected secondary results or the analysis population.

12. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Deaths

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 5% reporting threshold.

Most frequent non-serious adverse event terms

The safety summary reports no serious adverse events in either treatment group and no deaths. The denominators for serious adverse events are 36 for AFOLIA and 34 for Gonal-f® RFF. These are descriptive counts in the registry record and do not, on their own, establish that the treatments have identical safety profiles.

Other adverse events are reported for 21 participants in the AFOLIA group, with 36 at risk, and 22 participants in the Gonal-f® RFF group, with 34 at risk. These counts should be read alongside their respective denominators. They are not a formal comparison adjusted for the crossover design, exposure, or any other factors. The registry does not provide the event terms in the summary, and the list of top serious event terms is empty.

In general, a zero count of serious events in a study does not show that the risk is absent; it means that none were recorded in the reported group during the observed study. The registry does not give enough information in these summaries to evaluate event timing, severity categories for non-serious events, or a statistical comparison. This is a description of the posted safety data, not a medical recommendation.

13. What the Result Does, and Does Not, Mean

The primary pharmacokinetic result is a reported geometric least-squares mean ratio of 164.96 for AFOLIA relative to Gonal-f® RFF, with a 90% confidence interval from 137.82 to 197.45. The interval is above the stated 80%-125% equivalence margin. Accordingly, the posted primary result does not satisfy the equivalence objective described in the registry for baseline-corrected FSH AUC(0-last).

That conclusion is limited to the specified pharmacokinetic endpoint and the analysis reported. It does not establish whether either product is clinically preferable, and it should not be translated into advice about treatment. The trial was designed around single subcutaneous applications in healthy volunteers, with pharmacokinetic sampling after dosing. It does not directly answer questions about clinical outcomes, repeated use, or populations not represented by the study description.

Equivalence is a defined statistical claim: the estimated relative effect must be sufficiently precise and lie within the prespecified acceptable bounds. A result outside those bounds does not by itself explain why the estimates differ, nor does it establish a mechanism. The registry reports an arm-level geometric mean of 18.96 for AFOLIA and 10.47 for Gonal-f® RFF, but group summaries do not explain the individual variation or the reasons for it. The full ranges are descriptive, not measures of the uncertainty around the ratio.

Several contextual details remain unavailable in the registry record, including the complete statistical model, multiplicity strategy, missing-data methods, and the full analysis-set definition. Those omissions do not change the reported ratio and interval, but they limit independent evaluation of model assumptions and robustness. The registry lists secondary pharmacokinetic outcomes, yet their existence does not imply that their findings support or contradict the primary result; each outcome requires its own reported estimate and uncertainty.

The safety figures are also descriptive. The record reports no serious adverse events and no deaths, while other adverse events were recorded in both groups. These observations are not a comprehensive safety comparison. Overall, the result answers a focused pharmacokinetic equivalence question in the studied setting and should not be broadened beyond that scope.

14. Why This Trial Matters Statistically

This trial illustrates why the type of comparison matters. A conventional difference test and an equivalence analysis ask different questions. Equivalence testing evaluates whether the estimated relative effect is sufficiently close to the reference, according to prespecified bounds. The registry states an 80%-125% margin for AUC and reports a ratio with a 90% confidence interval. Reading the interval against the margin gives a direct interpretation of the stated objective.

The crossover design is another important statistical feature. Because participants received both treatments in an assigned sequence, comparisons can use within-participant information. This design can be efficient for pharmacokinetic work, where repeated concentration measurements are made after each treatment period. It also makes the analysis model and treatment of period, sequence, and possible carryover relevant. The registry describes the sequences but does not report the complete model, so readers cannot fully assess those analytical details.

The record also demonstrates why analysis populations and denominators need attention. Enrollment, pharmacokinetic endpoint summaries, and safety summaries have different recorded counts. Without an account of the reasons, readers should retain the denominators attached to each specific result rather than treating them as one common analysis group.

Finally, the distinction between a full range and a confidence interval is important. The arm ranges describe observed variability; the interval around the treatment ratio describes uncertainty in the comparative estimate. They are not interchangeable. The endpoint's geometric summaries and ratio are suited to a relative pharmacokinetic question, while the missing multiplicity and missing-data details limit assessment of the wider analysis program. These are general interpretive lessons drawn from the design and reported measures, not claims about outcomes beyond the registry record.

15. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

The study concerns healthy volunteers and single subcutaneous applications, so the registry record does not establish how the findings apply to other populations or dosing contexts. It focuses on pharmacokinetics rather than clinical outcomes. The crossover sequences are described, but the statistical model used for the reported comparison is not reported in the registry record. Details of any adjustment for period or sequence and assessment of carryover are also not reported.

The primary endpoint population description is incomplete: it states that the Pharmacokinetic Analysis Set includes subjects who received active study drug and had at least one measured, valid scheduled concentration, then ends with an unfinished additional criterion. The full analysis-set definition is not reported in the registry record. The record also does not explain why the primary pharmacokinetic summaries and safety summaries have different group counts.

The registry does not report a sample-size justification, missing-data methods, interim analyses, or a complete statistical analysis plan. It lists several outcomes and analyses, but the multiplicity strategy is not reported in the registry record. For another endpoint, the record states that values were missing or not estimable for a two-arm comparison without identifying the endpoint or explaining the cause.

These limitations restrict evaluation of prespecification, missingness, model assumptions, and the robustness of results beyond the posted primary estimate. The reported interval can still be compared with the stated equivalence margin, but the available record does not resolve these broader methodological questions.

16. Related Tutorials

17. Related Statistical Calculators

18. Sources

• ClinicalTrials.gov: Comparative Pharmacokinetics of AFOLIA and US Gonal-f® RFF Redi-ject After Single Subcutaneous Application — NCT02459418.
• Registry record last updated 2018-06-29; status last verified 2018-05.
• Results first posted 2018-05-29.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

19. Record Summary

NCT02459418 is a completed, randomized, open-label, phase 1, two-way crossover pharmacokinetic study sponsored by Fertility Biotech AG. It enrolled healthy volunteers and compared single subcutaneous applications of AFOLIA and US Gonal-f® RFF across two treatment sequences.

The primary endpoint was baseline-corrected FSH AUC(0-last). The sponsor-reported geometric least-squares mean ratio was 164.96, with a 90% confidence interval of 137.82 to 197.45. The interval lies above the stated 80%-125% equivalence margin, so the posted primary result does not meet the stated equivalence criterion. The record also lists secondary FSH and 17ß-estradiol pharmacokinetic outcomes and descriptive safety counts, while leaving several analysis details and missing-data explanations unreported.