Age associations with tacrolimus and mycophenolic acid pharmacokinetics in stable Black and White kidney transplant recipients: Implications for health inequities

Abstract Tacrolimus (TAC) and mycophenolic acid (MPA) provide maintenance immunosuppression and is dosed empirically in elderly kidney transplant recipients (KTRs) resulting in health inequities. Limited immunosuppressive pharmacokinetics are available comparing adult ages. This secondary analysis compared TAC and MPA pharmacokinetics and adverse effects (AEs) among young, middle‐aged, and elderly Black and White KTRs. The 12‐h TAC and MPA pharmacokinetics with AE evaluation were conducted in 67 stable KTRs greater than or equal to 6 months post‐transplant. TAC regimens were adjusted to target troughs. MPA regimens were adjusted using clinical response. Participants were: young: less than or equal to 40 years; middle age: greater than 40 to 60 years, and elderly greater than 60 years. Noncompartmental pharmacokinetic analysis determined area under the concentration‐time curve 0–12 h (AUC0‐12h), clearance (CL), and CL/body mass index (BMI) with 0‐h troughs. MPA enterohepatic recirculation (EHR), MPA‐AUC6‐12h/MPA‐AUC0‐12h, and MPA glucuronide (MPAG)‐AUC0‐12h/MPA‐AUC0‐12h were determined. Univariate analysis of variance (ANOVA) was conducted using SAS version 9.4. No group differences were noted for estimated glomerular filtration rate, MPA, and TAC doses. EHR was reduced in elderly with decreased MPA‐AUC6‐12h/MPA‐AUC0‐12h (p = 0.049) and increased MPAG‐AUC0‐12h/MPA‐AUC0‐12h (p = 0.036). MPA troughs (p = 0.045) were reduced in the elderly. TAC CL/BMI (p = 0.043) was reduced in the elderly. For therapeutic MPA AUC0‐12h: 30–60 mg·h/L, 34.3% KTRs achieved this target with 55.2% greater than the therapeutic range. 77.6% KTR were in the TAC AUC0‐12h target: 100–190 ng·h/mL and 19.4% were below this range with no age relationship. In 44% young, 26% middle‐age and 7.8% elderly subjects achieved target AUC0‐12h for both medications (p = 0.036). Neurologic AEs were manifested in the elderly (p = 0.014). Immunosuppressive pharmacokinetics demonstrated age‐related differences with reduced TAC CL/BMI and MPA EHR and increased neurologic AE in the elderly. This immunosuppressive regimen may require age‐adjusted individualization to optimize allograft function.


INTRODUCTION
Kidney allograft survival requires long-term immunosuppression. Tacrolimus (TAC), a calcineurin inhibitor and mycophenolic acid (MPA), an anti-proliferative, is the most common regimen prescribed in adult transplant programs in the United States. [1][2][3][4] Although short-term allograft survival has improved, chronic graft survival in Black recipients is reduced compared to other races. 1,2 Pre-and post-transplant factors that may account for poorer long-term graft survival include suboptimal medication adherence, immunosuppressive pharmacokinetic and pharmacodynamic differences, genetic variance, and donor-recipient mismatches, with racial differences in immunologic responses. 5,6 Elderly kidney transplant recipients (KTRs), who were rarely transplanted 30 years ago, may receive older, less functional organs but have improved long-term survival compared to age-matched counterparts on dialysis. 7 However, greater than 50% of mortality in older recipients is associated with cardiovascular disease, infections and malignancies, possibly related to empiric prescribing of immunosuppressive regimens that lacks individualization and age-adjusted dosing recommendations. [8][9][10] Increased age was found to be an independent risk for chronic allograft failure suggesting the need for age-adjusted dosing regimens during minimization of calcineurin inhibitors or glucocorticoids and to avoid drug-associated nephrotoxicity. 7,8 In addition, overimmunosuppression and severe adverse effects (AEs) commonly result in elderly recipients receiving medications with no age adjustment for declining hepatic and/ or renal function. [11][12][13][14] With the increased kidney allograft survival of middle-aged transplant recipients who survive beyond 60 years, it is imperative that age-adjusted immunosuppressive dosing be developed. 1,8,12 Post-transplant obstacles in elderly recipients are attributed to reduced allograft function, long-standing comorbidities, greater immunosuppressive-associated AEs, immunosenescence, and drug-drug interactions. 8,9,11,12 A National Institutes of Health (NIH) report summarized the necessity for research comparing elderly and younger transplant recipients by race because this knowledge gap places these subpopulations at risk for poor allograft survival and increased morbidities resulting in health inequities in older recipients. 15 Limited investigations of TAC and MPA pharmacokinetics comparing young and old KTRs have been conducted within the first 6 months post-transplant where acute clinical fluctuations are differences with reduced TAC CL/BMI and MPA EHR and increased neurologic AE in the elderly. This immunosuppressive regimen may require age-adjusted individualization to optimize allograft function.

WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?
Tacrolimus (TAC) and mycophenolic acid (MPA) provide maintenance immunosuppression and are dosed empirically in elderly kidney transplant recipients (KTRs) resulting in health inequities. Limited immunosuppressive pharmacokinetics are available comparing adult ages. This secondary analysis compared TAC and MPA pharmacokinetics and adverse effects (AEs) among young, middleaged, and elderly Black and White KTRs.

WHAT QUESTION DID THIS STUDY ADDRESS?
This secondary analysis compared TAC and MPA pharmacokinetics and AEs among young, middle-aged, and elderly Black and White KTRs.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?
This secondary analysis reported age-related differences in the commonly prescribed TAC and MPA regimen in stable KTRs. Reduced TAC Clearance/BMI and MPA enterohepatic recirculation and increased neurologic AEs were reported in the elderly.

HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY AND TRANSLATIONAL SCIENCE?
This report suggests that TAC and MPA immunosuppressive regimen may require age-adjusted individualization to achieve therapeutic exposure and the benefits of this approach must be confirmed in a larger KTR population.
ongoing. [16][17][18] In contrast, maintenance immunosuppression entails a prolonged therapy to prevent the recipient's immunoreactivity to the kidney allograft. 1,2,4 Minimal pharmacokinetic investigations of TAC and MPA with AE assessment relative to age have been reported during maintenance immunosuppression. It is also important to age-stratify transplant participants according to chronologic ages that includes young, middle aged, and elderly subjects to avoid skewing pharmacologic outcomes and assist in development of dosing continuums as patients age. In addition, age-related achievement of therapeutic area under the concentration-time curve 0-12 h (AUC 0-12h ) ranges for TAC and MPA in stable KTRs have limited investigations during maintenance therapy and could objectively guide individualized dosing regimens using age, race, and sex adjustments in pharmacologic models. [19][20][21][22] Therefore, age-adjusted comparisons of immunosuppressive pharmacokinetic and AE responses in stable KTRs can address this knowledge gap and provide key insight into development of customized immunosuppression and reduce health disparities post-transplant. 10,23 The primary objective for this report was to conduct a secondary analysis of TAC and MPA pharmacokinetics in young, middle aged, and elderly stable Black and White KTRs from a published prospective, observational 12-h pharmacokinetic study of these immunosuppressives that investigated the impact of race and sex. 24,25 For the primary objective of this report, the pharmacokinetic parameters determined for TAC and MPA were AUC 0-12h , AUC 0-4h , 0-h trough (C 0h ), maximum concentration (C max ), oral clearance (CL) with body mass index (BMI)adjusted CL (CL/BMI) and inclusion of MPA AUC 6-12h , ratio of MPA AUC 6-12h to MPA AUC 0-12h and ratio of the metabolite, MPA glucuronide (MPAG) AUC 0-12h to MPA AUC 0-12h . Secondary objectives included: (a) evaluation of agestratified, extra-renal AE scores and (b) analysis of participants achieving the therapeutic AUC 0-12h targets for each immunosuppressive and when these parameters are combined according to age.

Study population
Sixty-seven stable male and female Black and White renal transplant recipients receiving TAC (Prograf; Astellas Pharma US) and MPA as enteric-coated mycophenolate sodium (ECMPS; Myfortic; Novartis) for greater than or equal to 6 months participated in a 12-h pharmacokineticpharmacodynamic study (Figure 1). Clinical stability was determined by physical examination, comprehensive metabolic panel, and complete blood count. TAC troughs had been adjusted using the range of 4 to 10 ng/mL through therapeutic drug monitoring. ECMPS was dose-adjusted based upon clinical response. Medication adherence and ethnicity for two previous generations was verified. Estimated glomerular filtration rate (eGFR) was calculated using the four-factor modification of diet in renal disease equation. 26 Subjects were stratified into groups based upon young, middle age, and elderly patients.
Inclusion criteria were: (1) greater than or equal to 6 months post-renal transplant; (2) age 25-70 years; (3) first or second deceased-donor or living allograft recipient; (4) receipt of the tacrolimus and MPA regimen for greater than or equal to 3 months and on the same immunosuppressive doses for greater than or equal to 7 days; (5) baseline eGFR greater than 30 mL/min/1.73 m 2 with no change greater than 20% from baseline eGFR during prior two clinic visits with clinical stability confirmed by nephrologist; (6) leukocyte count greater than or equal to 3000/mm 3 and hemoglobin greater than or equal to 8.0 g/dL. Exclusion criteria were: (1) infection or acute rejection within 2 weeks; (2) drugs interfering with TAC or MPA absorption; (3) cytochrome P4503A4/3A5 or P-glycoprotein inhibitors or inducers within 4 weeks; and (4) significant medical or psychiatric diseases that would limit participation.

Study procedure
This was a cross-sectional, open-label, prospective clinical pharmacology study in stable Black and White recipients stratified by young, middle aged, and elderly patients conducted at the University at Buffalo (UB)-Nephrology Division at the Erie County Medical Center (ECMC). UB Institutional Review Board approved the study (IRB# PHP0599703-4) which was conducted in accordance with the ethical standards for human subjects and the 1964 Helsinki Declaration. Upon enrollment, patients provided written consent.
Patients were at steady-state conditions for tacrolimus and ECMPS. Proton pump inhibiters, H 2 antagonists, and antacids were discontinued for the prior 36 h. Patients took immunosuppressives between 5:30 and 6:30 p.m. prior to the study, fasted, and abstained from caffeine and alcohol for 12 h prior to study. At 6:00 a.m., patients were admitted, vital signs documented, and an intravenous angiocatheter inserted. A 0 h sample (~15 mL) was collected prior to immunosuppressives for drug troughs and laboratory tests (ECMC Clinical Chemistry Laboratory). Oral study medications (single lot of TAC [Prograf] and ECMPS [Myfortic]) were administered at 7:00 a.m. Patients remained upright throughout the study.
Standardized low fat meals were provided after 4 h. Antihypertensives were administered after 1.5 h and nonimmunosuppressives after 4 h. Blood samples (12 mL) were collected at 0 h and 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 h after drug administration. Whole blood samples for TAC analysis were aliquoted within 30 min. Plasma was harvested for MPA and MPAG analysis after being placed on ice within 30 min. All specimens were stored at −70°C until analysis.

Adverse effect assessment
Patients were evaluated using a validated immunosuppressive AE rating system of 14 extra-renal AEs. 27,28 Nephrologists used physical examination, review of systems, laboratory results, and medication adherence assessment and assigned a ranked score of 0 (no AE), +1, +2, or +3 (severe AE; see Table S1). A ratio of the sum of AE scores to the maximum possible score was determined (AE ratio). Individual AEs were combined into four composite categories: gastrointestinal (GI) including vomiting, diarrhea, dyspepsia, and acid suppressive therapy; neurologic (headache, tremor, and insomnia); aesthetic (acne, skin changes, hirsutism, and gingival hyperplasia); and cumulative AE (sum of GI, neurologic, and aesthetic AE).

Assay methodology for tacrolimus
TAC troughs were analyzed within 24 h at the ECMC Clinical Laboratory using the ARCHITECT tacrolimus assay (Abbott), a chemiluminescent microparticle immunoassay. 24 The plasma samples obtained for pharmacokinetic analysis were analyzed in batches using the same assay technique. The lower limit of detection was 0.5 ng/mL and intraday assay variability was less than 7%. The calibration standard curve ranged from 1 to 30 ng/mL and quality controls (QCs) were 3.0, 12.0, and 25 ng/mL (Bio-Rad). The interday coefficient of variation (CV) for each QC was less than 4% and intra-day CV was less than 5%. Random selected troughs and peaks (N = 40 samples) were analyzed using a validated liquid chromatography/mass spectrometry (LC-MS/MS) assay that was conducted by a Clinical Laboratory Improvement Amendment (CLIA) certified external analytical laboratory and compared to the results from the ARCHITECT (Abbott Laboratories) F I G U R E 1 Select TAC pharmacokinetic parameters stratified by age groups. Open dots represent White subjects and closed dots represent Black subjects. (a) Represents C 0h achieved in each patient using the range of 4 (dotted line) to 12 ng/mL (dotted lines). (b) Depicts TAC AUC 0-12h relative to age using the TAC therapeutic AUC of greater than or equal to 100 ng.h/mL, and less than or equal to 190 ng·h/ mL represented by the dotted lines. 7,8 There were 77.4% (52/67) of all subjects within the TAC target AUC 0-12h guide of 100 to 190 ng·h/mL for recipients greater than 1 year post-transplant. Note that the TAC AUC 0-12h was less than 100 ng·h/mL in 19.4% of all subjects. (c) Depicts TAC CL/BMI for each group. Reduced CL/BMI was noted in the elderly group with overall p = 0.043 and pairwise difference noted between the middle aged and the elderly (p = 0.042). AUC 0-12h , area under the concentration-time curve 0-12 h; BMI, body mass index; C 0h , 0-h trough concentration; CL, clearance; TAC, tacrolimus.
TAC assay with excellent agreement (R 2 = 0.98). For the LC-MS/MS assay, the interday and intraday CVs were less than or equal to 5% at the low and high concentration QC.

Assay methodology for MPA
An LC-MS/MS assay was used for the simultaneous analysis of plasma MPA and MPAG with 5, 5-diphenylhydantoin and flumethasone as internal standards, respectively. 29 Standard curve concentrations ranged from 0.145 to 15.5 μg/mL for MPA and from 22.1 to 295.2 μg/mL for MPAG. The lowest limit of quantitation was 0.072 μg/mL for MPA and 11.1 μg/mL for MPAG. The relative standard deviation for intraday variation for MPA ranged from 1.6% to 2.7% and 2.87% to 6.54% for MPAG. The relative standard deviation of interday variation for MPA ranged from 2.3% to 4.5% and 3.8% to 7.5% for MPAG.

Pharmacokinetic analysis
From intensive 12-h serial sampling, pharmacokinetic parameters for TAC and MPA that were directly measured included AUC 0-12h , ng·h/mL, C 0h , ng/mL, and C max , ng/mL. Oral TAC CL L/h was determined using the ratio of dose to AUC 0-12h . AUC 0-12h was determined by the linear trapezoidal rule using noncompartmental methods (Phoenix WINNONLIN version 6.3; Pharsight Corp). The C 0h concentration was included in the analysis because this parameter reflects the observed drug administration and the time since study commencement. The TAC therapeutic exposure guide of 100 to 190 ng·h/mL was used for comparisons based upon time post-transplant. 19 Pharmacokinetic parameters for MPA and MPAG included the AUC 0-12h , C 0h , and C max . The dose equivalent MPA was utilized. Oral apparent clearance of MPA was calculated as the ratio of MPA dose equivalent to MPA AUC 0-12h . MPA clearances were adjusted for BMI to assess the impact of standardized body weights. CL and AUC 0-12h were determined by the linear trapezoidal rule using noncompartmental pharmacokinetic methods (Phoenix WINNONLIN version 6.3; Pharsight Corp). The MPA therapeutic target AUC range of 30 to 60 mg·h/L was utilized as a guide. 22

Statistical analysis
Descriptive statistics were computed for all categorical and numeric variables and summarized by young, middle aged, and elderly patients. Comparisons were made using one-way analysis of variance (ANOVA) and an extension of Fisher's exact test for continuous and categorical variables. Box-Cox transformations were applied as appropriate. The univariate association between outcome measures for TAC and MPA pharmacokinetic parameters and AE scores by age were evaluated using one-way ANOVA models, with pairwise comparisons using Tukey adjustment. A covariate adjusted analysis was conducted where the association between outcome measures of pharmacokinetic end points and AEs relative to age were evaluated while adjusting for demographic and laboratory characteristics that included: albumin, eGFR, sex, race, diabetes, prednisone, statin use, time post-transplant, and CYP3A5*3*6*7 metabolic composite, in a one at a time manner, using analysis of covariance (ANCOVA) models. The AE scores had similar covariate adjusted analysis as previously described with the addition of TAC and MPA pharmacokinetic parameters. The Cochran-Armitage trend test was used to evaluate the therapeutic AUC 0-12h target for TAC and MPA separately and when combined stratified by age.
All model assumptions (including distributional assumptions) have been verified graphically. Diagnostic plots assessed model fit and the need for data transformations. If data transformation was required, it was indicated in each table. Statistical outliers were defined as studentized residuals outside ±3. All analyses were conducted in SAS version 9.4 at a significance level of 0.05. 30

Patients
Sixty-seven renal transplant recipients (35 Black and 32 White women and men) completed this study with no differences in age and time post-transplant. Demographics and clinical characteristics are summarized in Table 1 with no differences. Albumin, liver function tests, and hematologic parameters were within the normal range for all patients. No group differences in TAC and MPA doses were found with lower doses noted in the elderly.

Tacrolimus pharmacokinetics
Comparison of TAC pharmacokinetic parameters between groups are summarized in Table 2. Figure 1a-c presents the target pharmacokinetic parameters stratified by age. Age was associated with reduced TAC CL normalized for CL/BMI (overall p = 0.043) in the elderly with pairwise comparison noted between the middle age and elderly groups (p = 0.042). The elderly exhibited an approximate 30% reduction in CL/BMI compared to other groups and is depicted in Figure 1c. In 94% of subjects, the target C 0h was achieved due to therapeutic drug monitoring as depicted in Figure 1a. Note that 77.6% of subjects irrespective of age were within the target AUC 0-12h range of 100 to 190 ng·h/mL with 19.4% below the lower target range (see Figure 1b). Table 2 summarizes MPA pharmacokinetics with selected MPAG parameters to describe the metabolite to parent relationship over age groups. Figure 2a-c depicts the C 0h , AUC 0-12h and CL stratified by age. An age-related decline in MPA C 0h was found (p = 0.045) with pairwise comparison noted between the young and elderly groups (p = 0.035). No routine MPA therapeutic drug monitoring was conducted with troughs in the young and middle age groups exceeding the upper target. Enterohepatic recirculation (EHR) and its contribution to overall MPA AUC 0-12h was characterized using MPA AUC 6-12h /MPA AUC 0-12h (p = 0.049) and MPAG AUC 0-12h /MPA AUC 0-12h (p = 0.036) with pairwise differences noted between the young and elderly patients.

MPA pharmacokinetics
A decline in MPA AUC 6-12h /MPA AUC 0-12h and an increase in the metabolite ratio of MPAG AUC 0-12h /MPA AUC 0-12h in the elderly was found.
Although no age differences were found for total MPA AUC 0-12h , drug exposure comparisons were depicted in Figure 2b using the therapeutic range of 30 to 60 mg·h/L established for steady-state mycophenolate mofetil and cyclosporine regimens. This is further described in the legend for Figure 2 with ~34% of all subjects achieving the target MPA AUC 0-12h . Table 3 summarizes the number and percentage of subjects that achieved the therapeutic target AUC 0-12h for TAC or MPA by age and when immunosuppressives are combined. 19,20 Figure 3 depicts the percentage of these subjects within the therapeutic AUC 0-12h for each immunosuppressive and when combined. No differences were found between age groups for each immunosuppressive AUC 0-12h with a nonsignificant exposure decline noted in the elderly T A B L E 1 Demographics by age groups (n = 67). group. When subjects were combined, 34.4% of subjects were within the target MPA range with 55.2% above the upper target of 60 mg·h/L. For the TAC AUC 0-12h target, 77.6% of subjects were within the therapeutic range with 19.4% of subjects below the target range of 100 ng·h/mL. When the percent of subjects that achieved target AUC for both immunosuppressives depicted in Figure 3 and Table 3, only one elderly subject attained the target AUC 0-12h for both immunosuppressives (p = 0.036).

Adverse effect evaluation -neurologic
The immunosuppressive-related AEs are summarized in Table 3 according to GI, neurologic, aesthetic, and cumulative scores as described in the Methods. 27 The neurologic AE domain that included: tremors, headaches, and insomnia had a higher score in the elderly (p = 0.014) when adjusted for TAC AUC 0-4h . Pairwise trend was found between the middle aged and elderly groups (p = 0.070). All other AE domains were not significant (Table 4).

DISCUSSION
This novel report used secondary analysis stratified by age using a single center, prospective, intensive sampling study design that determined concurrent MPA and TAC pharmacokinetics with evaluation of validated extra-renal AE scoring in stable Black and White KTRs. All participants were enrolled using strict inclusion and exclusion criteria and were studied during maintenance immunosuppression at steady-state conditions with intensive sampling strategy. This analysis reported reduced TAC CL/BMI in the elderly compared to the middle aged with no age differences found with AUC 0-12h , doses, or trough concentrations in these stable KTRs. The reduced TAC CL may be supported by the declining hepatic function and structural changes described in the aging liver and during concurrent diseases. 13,14,31 These results may be further supported by a clinical pharmacogenomic study that reported higher dose/body weight normalized TAC troughs in the elderly compared to the young and middle aged KTRs with age and CYP3A5 genotype as notable covariates and suggested reduced metabolic clearance. 32 However, longitudinal TAC pharmacokinetics studies within the first 6 months post kidney transplant found no differences in dose normalized AUC 0-12h between the young and old recipients, which conflicts with our findings. 16 In our study, TAC therapeutic drug monitoring was used routinely in all participants and higher neurologic AE scores were found in the elderly when covariate adjusted TAC AUC 0-4h analysis was used. The increase in neurologic AE may contribute to increased morbidity post-transplant, which impacts the quality of life in the elderly. [10][11][12] In regard to MPA pharmacokinetic alterations over age, reduced EHR was found in the elderly compared to the younger counterparts that was quantitated by the increased ratio of the metabolite MPAG AUC 0-12h to the active moiety, MPA AUC 0-12h , and further substantiated by the reduced ratio of MPA AUC 0-6h to MPA AUC 0-12h . Minimal pharmacologic investigations into the age-associated changes in enterohepatic circulation and glucuronidation have been published and the general overviews summarize that no important changes occur with phase II metabolism over adult ages. 11,12,31 This is an important pharmacologic knowledge gap that is essential to address. Another important finding pertaining to the empiric MPA dosing in the combined groups of KTRs resulted in MPA AUC 0-12h and troughs F I G U R E 2 Select MPA pharmacokinetic parameters stratified by age groups. Open dots represent White subjects and closed dots represent Black subjects. (a) Represents C 0-h trough achieved in each patient using the target range of 1.9 mg/L to 3.5 mg/L (dotted lines). (b) Depicts MPA AUC 0-12h stratified by age with 34% (23/67) of the combined groups were within the therapeutic range of 30 to 60 mg·h/L. No MPA therapeutic drug monitoring was employed. Approximately 55% of all subjects exhibited MPA AUC 0-12h that exceeded 60 mg·h/L that included 56% (9/16) of young; 53% (20/38) of middle aged, and 61.5% (8/13) of elderly subjects. (c) Represents the apparent MPA CL which exhibits a fivefold variation in the combined patient groups. No age-related difference was found among these groups. AUC 0-12h , area under the concentration-time curve 0-12 h; C 0h , 0-h trough concentration; CL/F, total apparent clearance; MPA, mycophenolic acid.
T A B L E 3 TAC and MPA AUC 0-12h within the therapeutic target range individually and combined stratified by age. that exceeded the target ranges and may result in overimmunosuppression or increased AEs especially in the elderly recipients. 20,22 These MPA pharmacokinetic differences during maintenance therapy conflict with previous pharmacokinetic studies conducted in young and old kidney allograft recipients within the first 6 months posttransplant where no differences were found, and EHR was not described. 17,18 These pharmacokinetic findings for these commonly prescribed immunosuppressives provides evidence in support of developing systematic ageadjusted dosing regimens. An interesting age-related finding is the achievement of the therapeutic target AUC 0-12h range for either TAC or MPA and when groups are combined during maintenance immunosuppression. 19,22 See Table 3 and Figure 3. An interesting observation was that ~55% of the total subjects, regardless of age, had an MPA AUC 0-12 greater than 60 mg·h/L (Figure 2b), a value that exceeded the upper therapeutic MPA range. Less than 9% of these subjects had MPA AUC 0-12 less than 30 mg·h/L (lower target limit) regardless of empiric dosing. In regard to maintenance TAC with an established therapeutic target AUC 0-12h of 100 to 190 ng·h/mL for KTRs greater than 12 months posttransplant, 19.4% of the combined age groups exhibited AUC 0-12h below the lower target in spite of therapeutic trough concentrations. 19 Further prospective investigations are needed to determine the clinical importance of these AUC 0-12h values that are below and above the therapeutic range that have been established for TAC and MPA and relation to allograft outcomes based upon age stratification. When evaluation of the combined AUC 0-12h values was achieved for each of these immunosuppressives, a significant difference was found with only one elderly KTR achieving the target range for both drugs when compared to the young and middle age groups. This signifies that the maintenance immunosuppressive regimen may either not achieve an adequate therapeutic target in the older group or the elderly may require lower AUC 0-12h for each drug during maintenance therapy to sustain allograft function.

Subjects in therapeutic target
An intriguing finding was the manifestation of increased neurologic AE score in the elderly group when tacrolimus AUC 0-4h covariate adjustment was used in our statistical model. Associations of increased tremors have been reported with higher tacrolimus peaks, no data have been reported with age-related neurologic AEs. The AE assessment requires further investigation in a larger patient population stratified by age.
Some limitations exist in this report of the secondary analysis of TAC and MPA pharmacokinetics in stable Black and White KTRs that used a prospective, crosssectional, observational study design. The original subject enrollment did not use age stratification and was focused on race and sex enrollment targets. To address this issue objectively, we used covariate analysis added to F I G U R E 3 Relationship of stable KTR by age group that achieved TAC or MPA therapeutic AUC 0-12h target range. The bar graphs depict the percent of recipients by age group within the therapeutic AUC 0-12h target for MPA or TAC. This is nonsignificant for each immunosuppressive. When combining the patients that achieved both TAC and MPA target AUC 0-12h labeled "Both" in above graph for each age group, an age-related decline in achieving the target range was noted in the elderly (p = 0.036). AUC 0-12h , area under the concentration-time curve 0-12 h; KTR, kidney transplant recipient; MPA, mycophenolic acid; TAC, tacrolimus.
T A B L E 4 Immunosuppressive AEs rating by age. our statistical model. 30 Incorporation of clinical covariate analysis further identifies patients at risk for AEs or interpatient pharmacokinetic variability. In this secondary analysis, our statistical model also incorporated use of pairwise comparisons between patient groups to substantiate pharmacokinetic differences and AEs that may improve our understanding of age-related pharmacologic differences of these immunosuppressives during maintenance treatment.

CONCLUSION
Age-related differences in TAC and MPA pharmacokinetics were described with increased neurologic AEs manifested in this cohort. The elderly had slower TAC CL/BMI and reduced EHR of MPA. A higher neurologic AE score was also found in the elderly. To achieve health equity and optimize allograft survival in elderly transplant recipients, TAC and MPA immunosuppression may require age-adjusted individualization to achieve therapeutic exposure and the benefits of this approach must be confirmed in a larger patient population.