Early Metabolic Endpoints Identify Persistent Treatment Efficacy in Recent-Onset Type 1 Diabetes Immunotherapy Trials Free

Over the last 15 years, the Type 1 Diabetes TrialNet Study Group has conducted six immunotherapy clinical trials in participants who had recent-onset stage 3 type 1 diabetes, having the aim of preserving residual β-cell function (1,2). These trials consisted of nine treatments compared in a blinded and randomized manner with placebo. The mean 2-h mixed-meal tolerance test (MMTT) C-peptide area under the curve (AUC) at 12 or 24 months of follow-up has been the primary end point for all such clinical trials; specifically, the measure used is Ln(mean C-peptide AUC + 1), which is referred to throughout this text as the standard C-peptide AUC measure (3,4). This approach, however, does not 1) identify early therapeutic efficacy, resulting in more time- and resource-intensive trials, 2) consider timing of insulin or C-peptide secretion during the MMTT, or 3) consider the dynamics of the bidirectional relationship between glucose and C-peptide.

Here, we hypothesized that persistent treatment efficacy can be detected earlier than 12 months postintervention and that additional metabolic indices could detect this response. To this end, we assessed several metabolic measures to determine how well they distinguished participants in the treated and placebo groups and predicted the 12-month standard C-peptide AUC measure. For this analysis, we used stimulated metabolic measures of glycemia and β-cell function from MMTTs that include glucose, C-peptide, or both.

TrialNet recent-onset type 1 diabetes clinical trials were randomized, placebo-controlled, double-blind studies and included the following therapies: mycophenolate mofetil (MMF) alone and MMF in combination with daclizumab (DZB) (TrialNet MMF-DZB study TN02) (5), rituximab (TrialNet Anti-CD20 study TN05) (6), glutamic acid decarboxylase (GAD)-alum in two or three doses (TrialNet GAD study TN08) (7), abatacept (TrialNet Abatacept study TN09) (8), canakinumab (TrialNet Canakinumab study TN14) (9), and low-dose antithymocyte globulin (ATG) and ATG in combination with granulocyte-colony stimulating factor (G-CSF) (TrialNet ATG-GCSF study TN19) (10,11). Based on whether each intervention met its primary end point (reviewed in Table 1), trials were classified as either successful or unsuccessful, and they were analyzed in aggregate and individually.

Data from all TrialNet recent-onset stage 3 type 1 diabetes clinical trial participants (including placebo-matched control participants) who had complete MMTT data at baseline and follow-up were analyzed. Standardized MMTTs were conducted as previously reported, with the recent addition of automated insulin delivery devices being set to manual mode prior to the test (1,4,5). Baseline and stimulated glucose and C-peptide collected at 0, 30, 60, 90, and 120 min during the MMTT were evaluated.

Treatment arms (active vs. placebo) for each metabolic end point were compared via general linear models, both unadjusted and adjusted for age at trial entry, sex, and BMI. Metadata collected for each model included the R² as well as treatment arm P value and t or F value to identify interventions with high efficacy. The F value, or F statistic, can compare the variance of the group means and the within-group variances, allowing for a rank order. The R² determination was adjusted for age and baseline standard C-peptide AUC value.

Metabolic end points, as well as the change from baseline, were analyzed at the following visits: 3, 6, 12, and 24 months. Nine months was not included, as not all trials performed MMTTs at this time point. Measures of glycemia and insulin secretion and combined measures were included (Supplementary Table 1), specifically stimulated glucose and C-peptide values at 30, 60, 90, and 120 min, as well as early glucose or C-peptide response (difference between 30- and 0-min values), peak C-peptide, AUC measures, and the following four combination measures.

• 1.

  AUC ratio. The AUC ratio is calculated as the ratio of AUC C-peptide to AUC glucose. The 2-h AUCs for C-peptide and glucose were calculated using the trapezoidal rule, computed by dividing the AUCs by 120 min, the duration of the MMTT. A decrease in the AUC ratio indicates reduced insulin secretion as corrected for glucose, thus a worsening of metabolic function (12).

• 2.

  Index60. Index60 was developed as a pre-type 1 diabetes measure and is calculated using the following formula: 0.36953(log fasting C-peptide [ng/mL]) + 0.0165 ∗ glucose at 60 min (mg/dL) − 0.3644 ∗ C-peptide at 60 min (ng/mL) (13). An increase in the value is a sign of worsening metabolic function.

• 3.

  Centroid ratio. The centroid refers to the central point of the glucose and C-peptide response curve (GCRC), which is the shape made by connecting the mean 30-, 60-, 90-, and 120-min glucose and C-peptide values from the MMTT plotted on a two-dimensional grid with C-peptide on the x-axis and glucose on the y-axis (as has been done using oral glucose tolerance test [OGTT] data previously [14]). Several studies have denoted high correlation between OGTT and MMTT values (15,16). The C-peptide and glucose coordinates that define the centroid location are used to form the centroid ratio (C-peptide coordinate to glucose coordinate). A decrease in the measure is indicative of worsening metabolic function.

• 4.

  C-peptide index. We used the formula used in OGTT analyses for calculation of the C-peptide index, an indicator of first-phase insulin response, defined as the change in C-peptide from 0 to 30 min divided by the change in glucose from 0 to 30 min (17).

We refer to measures performed one time during the follow-up period as static measures, whereas we refer to changes in measures from baseline to a time point during follow-up as dynamic measures. We used a general linear model and F values to rank the measures at 3 and 6 months that identified a treatment effect between treated and placebo arms. Additionally, general linear models were used to identify which early measures were predictive of the standard C-peptide AUC measure (Ln[mean C-peptide AUC + 1]) at 12 months (18,19). To visualize the effect of each treatment, GCRCs were plotted over time, with vectors connecting the central points (centroids) of the curves.

All data generated or analyzed during this study are included in this published article or in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Central Repository at https://repository.niddk.nih.gov/studies/trialnet/. The data sets analyzed in the current study are available from the corresponding author upon reasonable request.

Participant characteristics, including age, sex, race, and BMI, from all clinical trials are reported in Table 1, in addition to details on the immunotherapeutic regimens and primary trial outcomes. Trials and treatment arms were grouped by their success in meeting their primary end point. Successful trials (e.g., rituximab, abatacept, and ATG) were analyzed in combination for common themes and individually to identify if any one trial was driving the results in the aggregate or had a different effect on glycemia or β-cell function. Similarly, unsuccessful trials (i.e., MMF alone or with DZB, GAD-alum, canakinumab, and ATG-GCSF) were analyzed in aggregate and individually. Diabetes duration at time of enrollment was similar between all trials, with mean ± SD of 2.8 ± 0.6 months.

Change in the MMTT-stimulated 2-h AUC C-peptide was the primary end point assessed after 1 year of follow-up for the rituximab, GAD-alum, canakinumab, ATG, and ATG-GCSF trials and after 2 years of follow-up for the abatacept, MMF, and MMF-DZB trials. We first assessed the ability of the standard AUC C-peptide (Ln[AUC C-peptide + 1]) at the 3- and 6-month study visits to detect a treatment effect in the three successful therapies (i.e., rituximab, abatacept, and ATG) versus their respective placebo groups. Second, we assessed dynamic measures (i.e., change in the standard AUC C-peptide from baseline to 3 months [abbreviated as Δ_0–3standard C-peptide AUC] and from baseline to 6 months [abbreviated as Δ_0–6standard C-peptide AUC]) to detect a treatment difference.

In the aggregate of successful trials, the absolute difference in the static mean standard C-peptide AUC between treated (n = 150 participants) and placebo (n = 92) groups at 3 months was (mean ± SD) 0.018 ± 0.40 ng/mL (F = 0.023, P = 0.881) and at 6 months was 0.129 ± 0.44 ng/mL (F = 3.558, P = 0.060). Similarly, among the individual trials, absolute differences in static standard C-peptide AUC were not significantly different between treated and placebo groups at 3 or 6 months (Supplementary Table 2).

However, when Δ_0–3standard C-peptide AUC was used, there was a significant difference between treated (n = 146) and placebo (n = 90) groups in the aggregate successful trials (0.068 ± 0.22 ng/mL, F = 4.747, P = 0.030). This was observed individually in the abatacept trial (0.111 ± 0.24 ng/mL, n = 70 treated and 32 placebo participants, F = 5.338, P = 0.023) but not with rituximab (0.101 ± 0.20 ng/mL, n = 48 treated and 27 placebo, F = 3.800, P = 0.055) or ATG (0.004 ± 0.20 ng/mL, n = 28 treated and 31 placebo, F = 0.000, P = 0.997). Using Δ_0–6standard C-peptide AUC, we found the difference between treated (n = 150) and placebo (n = 88) in aggregate successful trials to be 0.146 ± 0.25 ng/mL (F = 18.059, P < 0.001), with 0.150 ± 0.26 ng/mL (F = 3.920, P = 0.051), 0.141 ± 0.22 ng/mL (F = 8.241, P = 0.005), and 0.158 ± 0.27 ng/mL (F = 4.732, P = 0.034) in individual trials of rituximab, abatacept, and ATG, respectively.

We then asked whether alternative metabolic measures could detect early differences in treated versus placebo subjects. From the list of measures compiled (Supplementary Table 1), we first studied those that included a single measure (i.e., C-peptide alone or glucose alone). We continued to assess the difference between the change (Δ) in a measure from baseline to 3 months and baseline to 6 months to detect a treatment effect, as these appeared more efficacious at early time points than static measures based on the above results.

For the successful trials in aggregate, we selected the top three C-peptide measures and the top three glucose measures determined by their relative F value (Supplementary Table 3). For C-peptide alone, the measures with the highest rank for Δ_0–3 were the 120-min C-peptide, the second-hour fraction of the C-peptide AUC, and the standard C-peptide AUC measure. For glucose measures alone, the measures with the highest rank for Δ_0–3 were the 90-min glucose, the second-hour fraction of the glucose AUC, and the entire 2-h glucose AUC. Similar measures were identified for Δ_0–6 apart from 60-min glucose replacing the 2-h glucose AUC.

Previous studies in high-risk, autoantibody-positive populations have supported the predictive power of combined C-peptide and glucose measures derived from OGTTs in identifying progressors to type 1 diabetes and responders to immunotherapy (20–24). We therefore postulated that combined measures from MMTTs, which are highly correlated with OGTTs (15), might similarly identify effective immunotherapies. We again used the F value to rank the top three of four combined C-peptide and glucose measures (Supplementary Table 1). For Δ_0–3, the top three were the centroid ratio, AUC ratio, and Index60, whereas for Δ_0–6, the top three were the AUC ratio, C-peptide index, and Index60.

To identify the most effective markers across these three categories (C-peptide only, glucose only, and combined measures), we grouped these 11 measures and compared the F values within each follow-up period (Δ_0–3, Δ_0–6, and Δ_0–12) (Fig. 1A and Supplementary Table 3). Of the top measures for Δ_0–3, the F value was highest for the centroid ratio (F = 13.105, P < 0.001). In comparison, the standard C-peptide AUC measure had the lowest F value (F = 4.747, P = 0.030). In contrast, for Δ_0–6 the F value was highest for the standard C-peptide AUC measure (F = 18.059, P < 0.001), whereas the 60-min glucose was the lowest (F = 7.199, P = 0.008). To further support the accuracy of our approach of using the change from baseline, we ranked C-peptide, glucose, and combined measures for Δ_0–12, and the standard C-peptide AUC measure ranked highest (F = 27.252, P < 0.001), as was expected, since this is the measure and time point upon which we are judging efficacy.

The change in relative efficacy (assessed via F value) of the top 11 measures over 3, 6, 12, and 24 months is depicted in Fig. 2. This demonstrates a relative effectiveness of each of these measures, against each other, to identify a difference between treated and placebo arms of the aggregate successful trials. While the centroid ratio had the highest performance at 3 months followed by the 90-min glucose and AUC ratio, other features, such as prediction of future efficacy and sample size needed for a trial using such an end point, must be determined for plausibility. As for 6 months and beyond, all measures are outpaced by the dynamic standard C-peptide AUC measure.

To evaluate any differences that may be driven by a single trial, we also looked at the F-value and P value for the top C-peptide, glucose, and combined measures for each of the rituximab, abatacept, and ATG trials individually. These results are presented for Δ_0–3, Δ_0–6, and Δ_0–12 in Fig. 1B. To summarize, the majority of early findings are driven by abatacept and ATG, with combination measures performing similarly between these two therapies. In comparison, the top static measures for each trial individually are shown at 3, 6, and 12 months (Supplementary Table 4).

No difference between treated (n = 228) and placebo (n = 132) was seen in the unsuccessful trials in aggregate for Δ_0–3standard C-peptide AUC (0.028 ± 0.26 ng/mL, F = 0.990, P = 0.320) or for Δ_0–6standard C-peptide AUC (0.029 ± 0.29 ng/mL, F = 1.382, P = 0.241) or in the individual trials. For Δ_0–6, only the 30- to 0-min C-peptide (F = 4.028, P = 0.046) measure was significant in the aggregate of unsuccessful trials, but this was not seen in any trial individually. Notably, the Δ_0–6standard C-peptide AUC reached significance for the MMF-DZB (n = 32 treated and 35 placebo, F = 4.927, P = 0.030) study, despite the negative 2-year primary end point. However, this significance was not apparent in the unadjusted model (P = 0.110), indicating a strong effect of age, sex, and baseline BMI.

Early treatment effect can be best visualized using GCRCs (illustrative example shown in Fig. 3A). As seen in Fig. 3, the directionality and magnitude of the line connecting the central points from baseline to 3 months and later clearly demonstrates the differences seen in successful (Fig. 3B; n = 252) and unsuccessful (Fig. 3C; n = 360) immunotherapy trials. In aggregate, this difference can be seen at 3 months, which is quantified by the centroid ratio difference between treated and placebo arms in the successful trials (0.003 ± 0.01, F = 13.105, P < 0.001). Individual successful trials were also plotted (Supplementary Fig. 1), as differences are expected between the therapies, not only because of their different mechanisms of action but also because of the frequency of administration and potential time to maximal effect. ATG is administered over 2 days, whereas rituximab is given as four doses over 1 month. Abatacept is administered as three loading doses over a month followed by monthly infusions for 2 years. The differences in top measures by trial are summarized in Supplementary Table 4. While visually the effect of abatacept at 3 months is less dramatic (Supplementary Fig. 1), potentially because of its use as a maintenance therapy as compared with an induction therapy, the Δ_0–3centroid ratio is significant for both ATG and abatacept (Supplementary Table 4).

While several dynamic measures can identify early treatment efficacy, we sought to determine if these measures predicted a lasting effect by assessing the strength of correlation of each measure with the standard mean C-peptide AUC values at 12 months using R². In regression models of all study participants (n = 627), both 3-month and 6-month dynamic measures had poor R² values. The top 3-month dynamic measures (centroid ratio, 90-min glucose, and AUC ratio) demonstrated R² of 0.03, 0.08, and 0.07, respectively (all P < 0.001). The top 6-month dynamic measures (standard C-peptide AUC, AUC ratio, and 120-min C-peptide) demonstrated R² of 0.25, 0.05, and 0.08, respectively (all P < 0.001). This prediction improved greatly when age and baseline standard mean C-peptide AUC were included in the model and demonstrated that 6-month dynamic measures had higher R² than 3-month dynamic measures. The top 3-month dynamic measures (centroid ratio, 90-min glucose, and AUC ratio) demonstrated R² of 0.57, 0.57, and 0.63, respectively (all P < 0.001). The top 6-month dynamic measures (standard C-peptide AUC, AUC ratio, and 120-min C-peptide) demonstrated R² of 0.80, 0.69, and 0.71, respectively (all P < 0.001).

The three successful new-onset trials using a 1- or 2-year end point were conducted with sample sizes ranging from 60 to 112 participants (6,8,10). We found that a 6-month trial with two-sided α of 0.05 and 0.80 power, detecting a difference of 100% from the change seen in all placebo-treated individuals, would require 52 participants using the Δ_0–3standard C-peptide AUC measure or 54 participants using the Δ_0–3AUC ratio. An effect size of >100% over placebo was seen only in the ATG trial using the AUC ratio end point.

Unfortunately, a 3-month trial would result in impractical sample sizes of 112–230 participants. Of note, the ATG trial did see effect sizes of >100% over placebo using the 3-month dynamic AUC glucose. For trials involving this therapy, a potential 3-month dynamic end point using the AUC glucose could be done with a total sample size of 112 participants. However, this is a twofold larger sample size than the original ATG trial.

The use of the standard C-peptide AUC end point at 12 or 24 months takes substantial time and resources to reach conclusions on clinical trials, especially for new, early-phase studies. With a growing number of interventions in the pipeline poised for testing, there is a need to identify innovative end points so that more efficient trials can be conducted and more interventions can be screened for initial efficacy and safety. An end point measure of value in this setting should include the ability to 1) differentiate treated from placebo across trials, 2) predict long-term efficacy (as measured by our current gold standard measure, Ln[mean AUC C-peptide + 1]), and 3) support similar or smaller sample sizes than are used in current trials. Our results have identified several potential early end point measures with tradeoffs of predictive ability and sample size.

Using our novel approach of evaluating a range of measures (C-peptide only, glucose only, and combination measures), we found that dynamic changes in such measures from baseline were more likely to detect a treatment effect early compared with static measures. The standard C-peptide AUC measure performed well as a 3- and 6-month dynamic measure, as did others, specifically the glucose AUC (over 3 months), the centroid ratio (over 3 months), and the AUC ratio (over 6 months). However, dynamic measures used over the first 3 months of the trials demonstrated greater variability among individual trials and lower long-term predictive ability, and they would require larger samples sizes than the original trials. Over a 6-month period, instead we found more consistent results across aggregate and individual successful trials that were not seen in the unsuccessful aggregate or individual trials. Additionally, more of the variance of the final 12-month standard C-peptide AUC could be explained by several 6-month measures.

There are nuances of interest regarding the use of such metabolic measures in these immunotherapy clinical trials. Differences seen for top measures in individual trials may be related to the frequency and duration of drug administration and/or the mechanism of drug action. While all three successful therapies studied had significantly more C-peptide at 1 or 2 years compared with placebo and demonstrated delayed but then parallel C-peptide loss over time, the degree of C-peptide preservation at 3 months may be expected to vary based on drug mechanism or frequency of administration. Specifically, certain broad cell types—B cells and T cells—are targeted early following rituximab and ATG therapy, respectively. Abatacept, on the other hand, prevents T-cell costimulation without markedly reducing cell numbers. Additionally, rituximab and ATG are given in a small number of doses at the beginning of therapy and then stopped, akin to an induction therapy. Abatacept was given as repeated loading doses followed by monthly maintenance doses that continued throughout the trial. This may explain differences in the GCRCs between trials. For therapies that may take an extended time to accrue efficacy, early end points would not be ideal. Further precision in end point use based on the type of immune therapy may be of benefit and requires further study, especially given the differences in metabolic end points found in the B-cell– and T-cell–targeting therapies.

Limitations of this analysis are the post hoc nature and that the treatment effect at 12 or 24 months was predetermined using the standard C-peptide AUC measure, which may bias the other measures. While different studies had different entry criteria, such as age-group and time from diagnosis, most studies were within 100 days of diagnosis. To account for differences in age-groups, we adjusted for age (as well as sex and BMI) and studied outcomes not only in the aggregate but also for individual trials. This exploratory analysis did not adjust for multiple testing and would require targeted validation studies, which are planned in other recent-onset type 1 diabetes clinical trials outside TrialNet. Future attempts at study design could consider a comparative effectiveness design to allow for a more accurate head-to-head comparison in the recent-onset type 1 diabetes population, as this has only been done in a post hoc manner (19). Another analysis to consider would be use of a machine-based learning model to predict these potential outcomes more accurately (25). This, however, would require a larger number of trials.

Another factor to explore for intertrial differences could be related to the specific β-cell function being restored (e.g., first-phase vs. second-phase insulin secretion), specifically glucose-insulin dynamics. This could be explored moving forward by obtaining glucose, C-peptide, and insulin at early time points (10 and 20 min) to allow oral minimal model calculations of β-cell function, insulin sensitivity, and their integrated product, the disposition index (26). This type of precision assessment could identify ideal metabolic markers among individual responders (versus those treated with the therapeutic who did not respond). Further, the failure of a therapy to demonstrate efficacy at an early time point does not mean that a subpopulation of individuals did not respond. Other biomarkers (e.g., immune and omics-based biomarkers) are needed to identify these subpopulations. Ultimately, various factors could be used together to create a more precise early picture of responders to various β-cell–protective therapies.

On the level of a clinical trial, early intermediate end points could allow earlier assessment of whether a given agent is worth pursuing. Such measures are vital to adaptive trial designs. On an individual basis, it might allow determination of whether a given approach is working for that individual, and, if not, allow consideration of an alternative approach. Participants may be more willing to participate in a shorter trial or one where they would be switched to an alternate therapy should they not show any sign of improvement at an early time point. In the field of type 1 diabetes prevention, this could be done using the approved teplizumab therapy as the alternate to which a participant is switched.

In summary, we identified several novel dynamic measures at early time points indicating a positive treatment effect in successful, but not unsuccessful, recent-onset type 1 diabetes immunotherapy clinical trials. While a 3-month trial would be ideal from a cost and time perspective, more reliable measures were found in a 6-month trial period. However, a tradeoff is necessary between the measure’s ability to predict the 12-month outcome and the sample size such a trial would require. We plan to validate these findings in an external data set as well as assess them in prevention trials and at the individual responder level. These trials will include not only shortened trials but also adaptive trials that have the potential to require fewer participants and fewer trials to test multiple therapies, which are important goals. While regulatory acceptance of such an early end point in immunotherapy clinical trials in type 1 diabetes is unclear, we recommend using these data to complete faster early-phase new therapy trials seeking to accelerate the progression of therapies from the bench to the bedside.

Acknowledgments. We acknowledge the support of the Type 1 Diabetes TrialNet Study Group, which identified study participants and provided samples and follow-up data for this study.

E.K.S. and M.A.A. are editors of Diabetes Care but were not involved in any of the decisions regarding review of the manuscript or its acceptance.

Funding. The Type 1 Diabetes TrialNet Study Group is a clinical trials network funded by the National Institutes of Health (NIH) through the NIDDK, the National Institute of Allergy and Infectious Diseases, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development through cooperative agreements U01 DK061010, U01 DK061034, U01 DK061040, U01 DK061042, U01 DK061058, U01 DK085465, U01 DK085453, U01 DK085461, U01 DK085466, U01 DK085499, U01 DK085504, U01 DK085509, U01 DK103180, U01 DK103153, U01 DK085476, U01 DK103266, U01 DK103282, U01 DK106984, U01 DK106994, U01 DK107013, U01 DK107014, UC4 DK106993, and contract no. HHSN267200800019C. The JDRF also provided funding to the Type 1 Diabetes TrialNet Study Group. This work is supported in part by the NIH/National Center for Advancing Translational Sciences Clinical and Translational Science awards UL1 RR024131, UL1 RR024139, UL1 RR024153, UL1 RR024975, UL1 RR024982, UL1 RR025744, UL1 RR025761, UL1 RR025780, UL1 RR029890, UL1 RR031986, UL1 TR001085, UL1 TR001427, UL1 TR001863, UL1 TR001082, UL1 TR000114, UL1 TR001857, UL1 TR000445, UL1 TR002529, UL1 TR001872, UL1 TR002243, and PO1 NIH AI-42288 and a general clinical research center award (M01 RR00400). L.M.J. (K08DK128628-01) and H.M.I. (K23DK129799) are funded by the NIDDK of the NIH. H.M.I. is also funded by the Doris Duke Charitable Foundation through the COVID‐19 Fund to Retain Clinical Scientists Collaborative Grant Program (grant 2021258), The John Templeton Foundation (grant 62288), and the Pilot and Feasibility Grant from the Indiana Center for Diabetes and Metabolic Diseases (P30DK097512).

The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the JDRF.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Authors Contributions. L.M.J. wrote the first draft of the manuscript and performed data interpretation. D.C. and B.N.B. performed statistical analysis. M.A.A., W.M., M.J.H., W.E.R., S.E.G., K.C.H., M.J.R., E.K.S., D.K.W., A.M., A.P., and P.A.G. edited, reviewed, and approved the final version of the manuscript. H.M.I. and J.M.S. conceptualized the study and performed the analysis, interpretation, and review of the manuscript. L.M.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Handling Editors. The journal editor responsible for overseeing the review of the manuscript was Matthew C. Riddle.