Safety, Tolerability, and Pharmacokinetic Study of Methylene Blue Following a 1 mg/kg Intravenous Dose in Healthy Adults: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Safety, Tolerability, and Pharmacokinetic Study of Methylene Blue Following a 1 mg/kg Intravenous Dose in Healthy Adults (NCT02478281), a na phase 1 study. The primary outcome is Area Under the Curve (AUC) From Time Zero to the Time of Last Quantifiable Concentration (AUC 0-t).

2. Clinical Question

This is a descriptive question about a dose given to healthy adults, not a comparative question about whether methylene blue performs better than another treatment or no treatment. Without a comparator, the study cannot estimate a treatment contrast. The focus is instead on the exposure profile following administration and on adverse events recorded during the study. The record does not report a numerical AUC estimate, its variability, or the individual concentration observations, so the magnitude and precision of exposure cannot be assessed from the registry information. The study is listed as Phase 1 and completed. Its findings, as represented in the record, should be read in the context of the specified healthy-volunteer population and the single-dose regimen, rather than assumed to describe repeated dosing or treatment in people with methemoglobinemia.

3. Trial Design

The registry describes a completed Phase 1, one-period, single-dose study of Methylene Blue Injection, USP in healthy adult volunteers. Enrollment is listed as 12. The intervention was a dose of 1 mg/kg given intravenously over approximately 5 minutes. The record lists methemoglobinemia and congenital methemoglobinemia as conditions, while the study population itself is described as healthy adults.

The study is recorded as single-group, with allocation listed as NA and masking as none. No comparator arm is described, and the record explicitly distinguishes the study from a randomized comparison. As a result, its design is suited to describing observations after administration, including pharmacokinetic measurements and adverse events. It does not support a direct comparison with an untreated group or another treatment group.

The primary endpoint is AUC 0-t, defined in the record as area under the concentration-time curve from time zero to the last quantifiable concentration. The listed sampling schedule includes a pre-dose sample and post-dose observations at 0.042, 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 16, 36, 48, and 72 hours. These sampling times indicate that the endpoint depends on serial blood concentration measurements. The record names the intent-to-treat population for the primary endpoint, but it does not describe the operational definition of that population or the handling of participants without evaluable measurements.

The registry reports 11 outcome measures and 0 analyses. It does not report a numerical result for the primary endpoint. Details about the statistical plan, sample-size justification, interim analyses, and procedures for handling protocol deviations are not reported in the registry record. The stated sample-size illustrations are planning calculations rather than a description of the study’s original design or observed power.

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: Of the 12 participants enrolled, all met eligibility requirements and were treated.

Overall Study

Analysis population for the primary outcome, as defined in the registry: Intent to treat

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: Intent to treat

6. Primary Endpoint

The registry lists the primary outcome as Area Under the Curve (AUC) From Time Zero to the Time of Last Quantifiable Concentration (AUC 0-t), measured over pre-dose, 0.042, 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 16, 36, 48, and 72 hours post-dose.

The primary endpoint is the area under the concentration-time curve from time zero to the last quantifiable concentration, labeled AUC 0-t. The record defines it as a measure of systemic drug exposure, obtained by collecting blood samples over time and measuring drug concentrations in those samples. The scheduled measurements span pre-dose through 72 hours post-dose.

AUC is used in pharmacokinetic studies because it represents exposure across the observed concentration profile, rather than the concentration at a single sampling time. Integrating information across multiple measurements can describe how much drug is present in the circulation over the period covered by the observations. The 0-t version ends at the last data point that can be quantified; it is not, by definition, exposure over an unobserved period beyond that point.

The record specifies a geometric mean as the endpoint summary and a geometric coefficient of variation as the dispersion measure. In general, a geometric mean summarizes positive values on a multiplicative scale, while a geometric coefficient of variation describes relative variability on that scale. These summaries are commonly useful when pharmacokinetic measurements are skewed, although the registry does not explain why these particular summaries were selected for this study.

No numerical AUC estimate or dispersion value is reported in the registry record. Therefore, the endpoint’s observed level, variability, and precision cannot be described from the record. The endpoint name and sampling schedule define what was intended to be measured, but they do not establish the result.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

The registry record describes a pharmacokinetic endpoint and gives the way it is summarized, but it does not report the analysis details or the endpoint result. The explanations below distinguish general statistical concepts from the methods explicitly described in the record.

What does AUC 0-t represent?

In general, an area under a concentration-time curve combines measurements taken at different times into a measure of exposure over the observed interval. AUC 0-t refers to the interval beginning at time zero and ending at the last quantifiable concentration. It relies on the concentration measurements collected across that interval; it is not a direct measurement of exposure after the final quantifiable observation.

The record identifies blood sampling before dosing and at multiple post-dose times through 72 hours. Serial sampling allows the concentration profile to be followed over time and provides the observations used to derive AUC. The record does not specify how the area was calculated, how concentrations below quantification were handled, or how missing samples would be treated. Those details are not reported in the registry record.

Why use a geometric mean?

In general, a geometric mean summarizes positive measurements using a multiplicative scale. This can be suitable for pharmacokinetic measurements when values are asymmetric or when relative differences are more meaningful than absolute differences. The geometric mean is not interchangeable with the arithmetic mean: the two summarize data differently, especially when values are skewed.

The registry names the geometric mean for AUC 0-t and the geometric coefficient of variation for dispersion. A coefficient of variation describes variability relative to the scale of the measurements, and its geometric form is associated with a log-scale summary. These statistics can help characterize exposure and between-participant variability. However, the record does not report their numerical values, a confidence interval, or the method used to estimate uncertainty. Consequently, readers cannot assess the actual variability or precision of the endpoint from the registry record.

Why can over-dispersion matter?

In general, over-dispersion means that observed variability is greater than a statistical model would expect under its assumptions. When a model underestimates variability, uncertainty can appear smaller than it is, and statistical tests or intervals may be misleading. Methods that account for over-dispersion can improve the alignment between model assumptions and observed data. This is a general statistical consideration; the registry does not report a model for this study or indicate whether over-dispersion was assessed.

For a pharmacokinetic endpoint summarized geometrically, the distribution of measurements and the scale used for analysis are relevant to uncertainty estimation. The registry does not specify a transformation, distributional assumption, or model. It would therefore be inappropriate to infer that a particular model or adjustment was used.

What does a confidence interval add?

In general, a confidence interval expresses the uncertainty around an estimated quantity under a specified statistical procedure. A narrower interval indicates greater precision under that procedure, while a wider interval indicates more uncertainty. An interval does not by itself establish clinical importance or remove limitations of the design. For this study, the registry record does not report a confidence interval for AUC 0-t, and it gives no numerical endpoint estimate around which an interval could be interpreted.

The reported event counts also have uncertainty, particularly in a small single-group study. General methods for intervals around proportions can describe uncertainty in an observed event frequency, but the registry record does not provide such intervals. No interval should be inferred from the counts alone in this explanatory account.

How should planning calculations be read?

The planning assumptions described for the sample-size illustrations use a two-sided significance level of 0.05, equal allocation between two arms, and a normal approximation. They define the effect to detect as a stated fraction of an observed effect. In general, such calculations ask how many participants per arm might be needed under assumed conditions to detect a specified effect at a chosen significance level and power. They are planning illustrations, not the number of participants observed in this study and not observed power.

Those assumptions are not the design described in the registry, which is single-group and has no randomized comparator. A two-arm planning scenario is hypothetical in relation to this trial. The calculations are also dependent on the observed effect used as their basis; without a reported primary AUC result, the registry record alone does not allow readers to judge that basis. Numerical planning outputs and the power values associated with them are not reported in the registry record.

More broadly, an analysis plan would normally specify the estimand, analysis population, handling of missing observations, treatment of protocol deviations, and methods for estimating uncertainty. The registry names an intent-to-treat population for the primary endpoint but does not report these additional details. No specific statistical model or inferential procedure should be assumed.

10. Multiplicity, Missing Data and Analysis Populations

The registry lists 11 outcome measures and 0 analyses. It names AUC 0-t as the primary endpoint, but does not provide its numerical result or a description of analyses conducted. The planned hierarchy among the listed outcomes, if any, is not reported in the registry record.

Multiplicity refers, in general, to the increased chance of a false-positive finding when many hypotheses or endpoints are tested. Statistical plans may address this by specifying a primary endpoint, controlling error rates, or treating additional outcomes as exploratory. Although this record identifies a primary endpoint, it does not report whether or how multiplicity was considered across the outcome measures. No adjustment method should be inferred.

The primary endpoint is associated with the intent-to-treat population. The record does not define that population further, report how many participants had evaluable AUC data, or describe exclusions from analysis. It also does not specify how missing blood samples or concentrations below quantification were handled. The presence of scheduled sampling times does not establish that every scheduled measurement was obtained for every participant.

In general, missing data can affect an estimate when the availability of measurements is related to the measured outcome or to participant characteristics. Analysis methods may use assumptions about why data are missing, but those assumptions and methods are not reported. The registry also does not report sensitivity analyses, interim analyses, or a statistical analysis plan. The extent to which these issues affected the endpoint result cannot be assessed from the registry record.

11. 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 registry reports 0 participants with a serious adverse event among 12 at risk in the Methylene Blue Injection, USP group. It reports 10 participants with other adverse events among 12 at risk, and 0 deaths. These are counts of affected participants as recorded, not a description of event severity or causality.

The record does not list serious adverse-event terms, and it does not identify the other adverse-event terms in the registry record for this account. It also does not report the timing, duration, recurrence, severity grading, or investigator assessment of relationship to treatment. The event figures therefore cannot answer which events occurred or whether a particular event was considered related to the dose.

In general, a zero count in a study does not establish that an event is impossible or that the risk is absent. Counts from a small single-group study provide limited information about uncommon events, and no comparator is available to contextualize the observed counts. The registry’s figures should be read as recorded observations in this study, not as a comparative safety estimate or a guarantee about safety in other populations or dosing settings.

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

This study is registered as a completed Phase 1 investigation of safety, tolerability, and pharmacokinetics after a single intravenous dose of methylene blue in healthy adults. The record specifies 1 mg/kg and an administration period of approximately 5 minutes. Its primary endpoint is AUC 0-t, a measure intended to describe systemic exposure across the observed concentration-time profile.

The record does not report a numerical result for that primary endpoint. It therefore does not establish the observed exposure level, its variability, or the precision of the estimate. Although the sampling schedule and summary statistics are named, those details describe the measurement plan rather than the result itself.

The adverse-event counts are descriptive: 0 participants with serious adverse events among 12 at risk, 10 with other adverse events among 12 at risk, and 0 deaths. These counts do not, by themselves, establish causation or permit comparison with another treatment. The event terms and additional clinical details are not reported in the registry record.

The study’s single-group design means it cannot estimate a randomized difference between methylene blue and a comparator. Its healthy-adult population also limits direct application to people with the listed conditions. A single-dose study does not describe repeated administration unless additional evidence is available; the registry record does not report such evidence.

Accordingly, the record supports a description of the study’s design, intended pharmacokinetic endpoint, and recorded event counts. It does not provide enough information to quantify AUC, assess its statistical precision, determine the cause of adverse events, or draw a comparative conclusion. These limits concern what can be concluded from the registry record, not whether the study may have generated additional information elsewhere.

13. Why This Trial Matters Statistically

Early pharmacokinetic studies connect a dosing regimen to measured drug concentrations over time. AUC 0-t is one way to characterize that exposure using serial measurements, and the record’s sampling schedule indicates that the endpoint depended on observations across the post-dose period. Identifying the endpoint and its intended summary is useful for understanding the study’s scientific question even when the numerical result is not available in the registry record.

The study also illustrates the distinction between descriptive evidence and comparative evidence. A single-group design can record what happened after administration, but it cannot provide a randomized contrast against another group. This matters when interpreting both pharmacokinetic summaries and adverse-event counts: without a comparator, observed values are not estimates of differences between treatments.

Reporting the analysis population, summary measure, dispersion measure, and sampling times helps readers understand the intended measurement framework. At the same time, the absence of the AUC estimate, uncertainty measures, and analysis details limits independent statistical interpretation. The registry lists 11 outcome measures but 0 analyses, so readers cannot determine from this record how the outcomes were analyzed or how conclusions were drawn.

The safety counts also show why event descriptions and context matter. Counts alone do not reveal event types, severity, timing, or attribution. In general, estimates are more informative when accompanied by clear definitions and uncertainty measures, especially when a study has a single group and a limited enrollment.

Finally, planning illustrations should be separated from the trial’s actual design. The stated hypothetical two-arm assumptions do not turn this one-group study into a comparative trial, and planning calculations are not observed power. Keeping those distinctions clear helps prevent a design description, an analysis result, and a future-study planning exercise from being treated as the same kind of evidence.

14. 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 registry record provides limited results for interpreting the primary pharmacokinetic question. It names AUC 0-t, its geometric summary, and its dispersion measure, but does not report the numerical estimate, dispersion value, or confidence interval. The magnitude and precision of systemic exposure therefore cannot be assessed from the record.

The study is single-group, with allocation listed as NA and masking listed as none. No control group is described, so the design cannot support a randomized comparison or provide a basis for separating changes or adverse events associated with dosing from other explanations. The record describes a single dose, not a repeated-dose regimen.

Enrollment is listed as 12, and the adverse-event group reports 12 at risk. The registry does not provide participant-level concentration data, the number with evaluable AUC measurements, or details of missing samples. It also does not explain how concentrations below quantification or incomplete profiles were treated. Those omissions matter for assessing the endpoint’s calculation and potential bias.

The adverse-event record gives affected-participant counts but not event terms, severity, duration, timing, or attribution. It reports no serious-event terms. The safety information is therefore limited to the stated counts and does not allow a detailed characterization of the events.

Further details about sample-size justification, statistical analysis methods, multiplicity, interim analyses, protocol deviations, and sensitivity analyses are not reported in the registry record. The stated planning assumptions describe a hypothetical two-arm calculation rather than this single-group design. These limitations restrict what can be concluded from the registry record and should not be filled in by assuming methods or results that are not documented.

15. Related Tutorials

16. Related Statistical Calculators

17. Sources

• ClinicalTrials.gov: Safety, Tolerability, and Pharmacokinetic Study of Methylene Blue Following a 1 mg/kg Intravenous Dose in Healthy Adults — NCT02478281.
• Registry record last updated 2025-06-05; status last verified 2018-01.
• Results first posted 2025-06-05.
• 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.

18. Record Summary

The registry record describes a completed Phase 1, single-group study of Methylene Blue Injection, USP in healthy adult volunteers. Enrollment is listed as 12. The intervention was a single intravenous dose of 1 mg/kg given over approximately 5 minutes, with no comparator and no masking.

The primary endpoint was AUC 0-t, intended to measure systemic drug exposure from time zero to the last quantifiable concentration. The record specifies a geometric mean and geometric coefficient of variation, but does not report a numerical endpoint result or its precision. It lists 11 outcome measures and 0 analyses.

For the methylene blue group, the registry reports 0 participants with serious adverse events among 12 at risk, 10 with other adverse events among 12 at risk, and 0 deaths. Event terms and further clinical details are not reported in the registry record. The single-group design supports descriptive interpretation only and does not provide a randomized comparison.