In the last decade, elderly (Eld) patients have been more frequently listed for renal transplantation.1 At our center, from 2007 to 2014, the percentage of renal transplant recipients older than 60 years increased from 11% to 21%.
The calcineurin inhibitor tacrolimus (TAC) is the most used calcineurin inhibitor in solid organ transplantation. Most features of the pharmacokinetics (PK) of TAC derives from studies in healthy volunteers and transplanted patients. However, all such studies included individuals with a mean age usually lower than 60 years. Because the world population and, proportionally, the transplanted population, is getting older,2 it seems reasonable to study TAC-PK in the Eld compared with younger controls (Ctrl).
Aging may modify drug absorption, metabolism, and drug clearance.3,4 There is evidence, for example, that the liver mass and liver perfusion decrease with aging.5,6 P-glycoprotein is a transmembrane protein that mediates the efflux of TAC from the enterocytes, lymphocytes and other cells. Aging may interfere with the expression and activity of this transporter.7 Tacrolimus is metabolized by the cytochrome P450 (CYP) 3A5 and the polymorphism of the gene encoding for CYP3A5 partially explains the large interindividual bioavailability of TAC.8 There is evidence that the activity of CYP decreases substantially with aging.3,4 Tacrolimus has also side effects, and particularly in the Eld, the decrease in insulin secretion, which is clearly related with TAC exposure9 may be very important.
The abovementioned comments per se justify an analysis on whether or not the PKs of TAC changes with aging.
In this study, we report the longitudinal evaluation of TAC-PK in Eld recipients, submitted to a renal transplantation under the regimen of TAC plus enteric-coated (EC) sodium mycophenolate (MPS) and steroids, when compared with younger Ctrls under the same immunosuppressive regimen.
Elderly patients participating in this study were those enrolled in the nEverOld study—Renal Transplantation in the Eld (NCT 01631058), a single-center, prospective randomized trial, comparing low TAC/Everolimus (EVL) with regular TAC/EC-MPS in Eld recipients (≥60 years at renal transplantation). The nEverOld trial and the sub-studies were approved by the institutional Review Board in research and were conducted in full compliance with Guidelines for Good Clinical Practices and Declaration of Helsinki, as required by our committee.
All patients enrolled in the TAC/EC-MPS arm as well those in the low TAC/EVL arm were studied but, in the latter case, before switching from EC-MPS to EVL, what usually occurred at a mean of 37 days. These patients performed only PK1 (day 7) and PK2 (day 30) measurements. In other words, in this specific TAC-PK study, only patients with TAC/EC-MPS were analyzed.
Control patients were those younger than 60 years transplanted at our center during the nEverOld enrollment period who accepted to participate in the TAC-PK studies. At the time of this analysis, 44 Eld patients were active in the nEverOld trial with 22 in the TAC/EC-MPS arm. Therefore, most of the 44 patients performed the TAC-PK studies at 7 and 30 days but only the 22 patients, in the TAC/EC-MPS arm, performed the analysis of 60 and 180 days.
Both groups received a single dose of thymoglobulin (Genzyme, Sanofi Company, Cambridge, USA) of 2 mg/kg intraoperatively. Generic TAC started with an initial oral dose of 0.1 mg/kg twice daily and adjusted to maintain the predose blood level (C0) between 5 and 10 ng/mL throughout the first 3 months and 3 to 8 ng/mL, thereafter. The generic TAC (Tarfic; Libbs Farmaceutica, Sao Paulo, Brazil) is approved according to the rules that define generic drugs by Cmax and area under the curve (AUC), as any other generic immunosuppressant,10 in a crossover study in healthy volunteers where the generic is compared with a brand name drug. The same generic TAC was maintained during the whole study to avoid interferences11 Oral prednisone starts at 0.5 mg/kg per day and was tapered to 0.1 mg/kg per day at the end of the first month. Enteric-coated mycophenolate sodium (Myfortic, Novartis, Basel, Switzerland) was started on the day of transplantation at an oral dose of 720 mg every 12 hours and adjusted according to side effects.
Tacrolimus PKs were scheduled in 4 consecutive 12-hour PKs at 7, 30, 60, and 180 days after transplantation. After an overnight fasting, blood samples were collected before and at 20, 40, 60, 90,120,180, 240, 360, 480, 600, and 720 minutes post-TAC oral dose. Each blood sample was saved into ethylenediaminetetraacetic acid tubes for determination of total TAC blood concentrations by ultraperformance liquid chromatography coupled to a mass-spectrometer repetition.
A breakfast was offered 2 hours after the TAC morning dose. Tacrolimus was given in conjunction with the other immunosuppressive drugs, prednisone, and EC-MPS.
Besides TAC blood concentrations in the PK curves, the other PK parameters calculated in this study were:
Tau (hours): time of the complete curve
AUC0-12hrs(ng*h/mL): area under the curve at dosing interval;
Tmax (hours): time of maximum concentration sampled during a dosing interval;
Cmax (ng/mL): maximum concentration;
Cmin (ng/mL): minimum concentration;
Cavg (ng/ml): area under curve from dose time to Tau divided by (dose time + Tau);
%_Fluctuation: = 100*(Cmax-Cmin)/Cavg, for Cmin and Cmax between dose time and Tau;
HL_Lambda_z (hours): apparent terminal half life;
Clss_F (L/hr/kg): an estimative of the total body clearance normalized by dose and weight of each patient (=Dose/AUC0-Tau).
Data were analyzed with the use of Phoenix WinNonlin version 6.3 for Windows (Pharsight Co., Monatain View, CA) as a noncompartmental distribution. Parametric data are expressed as mean ± SD.
Generalized Estimated Equations (GEE) were used to evaluate the effect of Tacrolimus Clearance and age (Eld vs Cntl), race (white vs black) at time points (D7, D30, D60, and D180). All the interactions were considered in the analysis and the post hoc Sidak Newman-Keuls was used to address the multiple comparisons needed for main and interaction effects. Besides age groups, sex, and donor source (live or deceased) were included at the GEE as covariates, since baseline differences were detected in group comparison. For all comparisons, the α error was set at 5% (P < 0.05), and the analyses were performed using SPSS v.19 software.
During the studied period two hundred eight 12-hour TAC-PK were available for analysis in the 44 Eld and 31 Ctrl kidney transplant recipients. The demographics of these groups are described in Table 1. Besides the age that was 30 years older in the Eld group, they also differed from each other in some other characteristics. The Eld group were more frequently white men who received more kidneys from deceased donors whereas Ctrl group had more female patients with a balanced race receiving more kidneys from live donors than the Eld group.
Table 2 shows some clinical parameters of both groups that could interfere with the TAC-PK. Elderly patients had an estimated glomerular filtration rate (eGFR) (by the Modification of Diet in Renal Disease-4 equation) statistically lower than Ctrl group at all times. In the same way, blood hemoglobin levels were lower in the Eld group but statistically significant only at 60 days. Serum albumin was, at any time-point, lower in the Eld group and statistically lower at periods 30 and 60 days. On the other side, liver enzymes and prothrombin time were very similar between groups. The prednisone daily dose that may alter the TAC-PKs12 was also similar in both groups.
Table 3 shows the PK parameters of both groups. Tacrolimus PKs were performed at 8 ± 2 days (day 7), 31 ± 4 days (day 30), 63±6 days (day 60), and 185±10days (day 180). Not all patients performed all the PK at all timepoints. The number in each group at a specific PK is shown at table.
Tacrolimus daily dose decreased throughout the study periods in both groups and was significantly lower in the Eld group at all timepoints. Tacrolimus dose, given before the PK study (half of the daily dose) and adjusted by body weight progressively decreased in both groups throughout time and was lower in the Eld group than in Ctrls for any period analyzed (Figure 1).
Unadjusted PK Parameters
In both groups, the TAC target therapeutic window was based in the trough level (Ctrough). As expected, there were no differences in Ctrough between the 2 groups throughout the study period but was lower at day 180, in both groups. In the same way, Cmax was not statistically different between groups. Cmax decreased in both groups at day 180. Cavg also did not differ between the groups. Tmax occurred between 1.3 and 1.6 hours and did not differ between groups at any time-points. Area under the curve at dosing interval decreased in both groups at day 180 and was not statistically different between groups. Tacrolimus apparent half-life was similar in both groups and did not change over time. Fluctuation was lower in Eld group during all timepoints analysis and statistically lower only at Day 60.
Figure 2 and Table 3 show TAC-PK curves adjusted (adj) by dose/body weight, in both groups at time-points and altogether (last column). The mean adjCmin was statistically higher in the Eld group compared with Ctrl in all timepoints analysis and statistically significant at days 7, 60, and 180. The mean adjCmax was statistically higher in the Eld group compared to Ctrl in all timepoints analysis and statistically significant at days 7, 60, and 180. The mean adjAUC0-12h was also higher in Eld group at all timepoints analysis and again statistically significant at days 7, 60, and 180.
Estimated total body TAC clearance was lower in the Eld group in all timepoints and statistically different at days 7 and 60.
As there were more white patients in the Eld group than in Ctrl group, an analysis of group and race interaction was performed by GEE for TAC estimated total body clearance. There was no interaction between race and age group in the TAC clearance in the multivariate analysis showing that the Eld group had a lower TAC clearance than Ctrl, independent from race (data not shown). When only white patients were analyzed, TAC clearance was again statistically lower in the Eld group compared with Ctrl group at days 7 (P = 0.01), 30 (P = 0.052), and 60 (P = 0.042) (data not shown). On the other hand, for the Afro-Brazilians, there were no statistical differences at days 7, 30, 60, and 180 although the number of patients was too small in both groups to draw any conclusion.
Due to the differences in sex between the Eld and Ctrl groups, we also analyzed this feature. In the Eld group, there were no differences in all TAC-PK parameters between male and female patients. However, TAC clearance was higher (P = 0.057) in the nonwhite Eld patients also showing a sex and race interaction (P = 0.002). Elderly white males had a reduced clearance as compared to Eld Afro-Brazilian men (0.43 ± 0.26 vs 0.87 ± 0.32 L/h per kg). Surprisingly, this difference was not found in female white patients compared to female Afro-Brazilians (0.64 ± 0.58 vs 0.60 ± 0.19) but the small sample size from this subset of patients were too small in both groups to have enough power to generalize this finding.
In the same way, because there were more deceased donors in the Eld group compared with the Ctrl group, we have also analyzed this interaction (age and kidney source). There is no difference in TAC estimated total body clearance for live or deceased donors (P = 0.760) and no interaction between age and kidney source (P = 0.178).
Figure 3 shows the correlation between TAC through level and exposure, evaluated by the area under the time-concentration curve. There was a high correlation between these 2 parameters with a R2 higher than 0.76 for both groups. The correlation was much higher in the TAC through-level range of 2 to 9 ng/mL, the therapeutic window usually used in renal transplantation. In this range, and starting from an exposure of 41 to 42 ng/h per mL at TAC through level of 1 ng/mL, for each 1 ng/mL increment in through level, AUC0-12h increases 16 and 18 ng/h per mL for Eld and Ctrl groups, respectively.
This study demonstrates the TAC-PKs in a group of Eld renal transplant recipients compared with a younger group. Our data show that Eld recipients need a lower TAC weight-normalized dose to achieve a higher weight-normalized exposure. This higher exposure is due to a lower TAC clearance in the Eld recipient.
The DeKAF investigators had already anticipated our findings of higher TAC trough levels in a larger (n = 374) cohort of Eld and young recipients enrolled in a multicenter trial.13 Similar to our findings, their data showed that older patients had a higher normalized TAC trough levels although receiving a lower weight-normalized dose. Nevertheless, they have analyzed TAC trough levels only. To our knowledge, this is the first comparison of longitudinal, full TAC-PK analysis in Eld recipients compared with a younger Ctrl.
In accordance with the United Nations agreement regarding definition of an old person,14 we defined Eld renal transplant recipients as those 60 years or older at the time of transplantation because the criterion of 65 years or older, usually used in developed countries, may not apply to developing countries. In our study, the Eld patients were, on average, 30 years older than the Ctrl group. In addition, 53% of our cohort was older than 65 years and 13% was older than 70 years. This range of ages is probably representative of this kind of Eld transplant population, because it is unlikely that many individuals older than 75 years will undergo renal transplantation in our center in the next decade.
Our Eld group differed considerably from the Ctrls in terms that they were mostly composed of white men receiving kidneys from deceased donors. This is exactly a sample of the Eld population we have been transplanting in the last few years.
Due to a higher frequency of deceased kidney donors (frequently of expanded criteria) in the Eld group, the eGFR was lower in the Eld recipients but again this range of GFR is not supposed to interfere with TAC-PK. A lower hemoglobin level in the Eld group could contribute to more free TAC levels and an increased in TAC metabolism, but this effect seems to be too low to interfere with TAC-PK.15 What is more, the increased free TAC levels would increase the TAC clearance but this rationale is the contrary of our findings of a decrease TAC clearance in the Eld group.
In this study, both groups received the same generic tacrolimus formulation, approved by the Brazilian health authorities in accordance to the international criteria of bioequivalence for generic drugs. Generic tacrolimus is provided, at no costs by the Brazilian health system to transplant recipients. This is important to mention because, although we, like others,16 have safely used generic tacrolimus, this is still a controversial issue.11,17 However, as both groups used the same TAC formulation and TAC levels were measured by the ultraperformance liquid chromatography coupled to a mass spectrometer repetition, that measures specifically the molecule of TAC, the findings here reported can certainly be extrapolated to other TAC formulations.
Our TAC-PK data, in the Ctrl group, are very similar to the published data of TAC-PK in transplanted patients at similar time after transplantation and equivalent ages17-20 which seems to demonstrate that our Ctrl group was adequate for comparison.
Notwithstanding, there is a lack of studies comparing PK of immunosuppressive drugs in Eld recipients compared with younger Ctrls.
Miura et al21 analyzed TAC-PK in 12 Eld (≥60 years) recipients 1 month after transplantation compared with 41 young recipients (20-39 years) and to a mature group (40-59 years) of 57 recipients and concluded for no influence of age on dose-adjusted TAC-PK. However, analyzing their data of the Eld and young groups only (because the mean age of our Ctrl group is similar to their young group) and compared with our data at 1 month, they have also found a lower TAC dose, a higher adjusted Cmax, a higher adjusted AUC0-12h and a lower TAC clearance. Nevertheless, contrarily to our data, none of those parameters achieved statistical significance. These concordant results with discordant statistical significance could be explained by their small sample size of Eld recipients (n = 12) and PKs performed (n = 12) compared with our data with 44 Eld recipients and 108 PKs. Therefore, our data as well as theirs are in accordance with a diminished TAC clearance with consequent higher dose-adjusted exposure in Eld recipients.
Some reviews are available regarding the possible changes age can do on the drugs PKs and two recent ones summarized them.5,6 It has been suggested that Eld people require a lower dose of many drugs but no definitive data has proven it for each particular drug.
The fact that total and functional hepatic flow reduces with age22 led to the assumption that reduced drug clearance could be the result of a diminished liver blood flow along with reduced liver mass23 ending with a diminished hepatic clearance. In our study, Eld recipients had a lower serum albumin level as compared with the younger Ctrls. Low serum albumin levels may be an indirect indication of a diminished hepatocellular mass. A liver function test, with radio-PK modeling of technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin, used to evaluate hepatocellular mass,24 showed a high positive correlation of liver mass and serum albumin.25 Therefore, the lower serum albumin level in our Eld cohort could be an indirect sign of reduced hepatocellular mass although we cannot prove this.
Other factors may also contribute. Drugs, such as, TAC and CyA, which are substrates of CYP enzymes may have their metabolism diminished with age due to a decrease in the activity of this enzyme system. Sotaniemi et al4 showed that the cytochrome P450 activity declined after 40 years (−16%) to a level that remained unaltered up to 69 years, and declined further (−32%) after 70 years. Also, the antipyrine (phenazone) clearance declined after 40 years by a rate of 0.34 mL/min per year (−29%) toward older age. In fact, the half-life of drugs, metabolized by the CYP, may increase up to 70% in adults older than 65 years.3
Also, transplant recipients with CYP3A5*1 allele require higher TAC doses than the CYP3A5 *3/*3 genotype.8,21,26 Unfortunately, we have not determined the CYP3A5 polimorphism and activity in our population to evaluate this aspect.
We acknowledge that the differences in race with more African-origin patients in the Ctrl group could introduce a bias to our results. For example, African Americans have lower TAC trough levels than whites possibly due to the fact that they more frequently have the CYP3A5*1 allele.27 Age and the CYP3A5*1 genotype have a large effect on TAC trough level13 and in countries where race is well differentiated among patients, African American patients require higher doses of TAC and cyclosporine A.28,29 However, definition of patients' race in Brazil is difficult due to a mixture of racial origins existing in almost every patient and because each patient, by the law, defines its own race.
However, when we analyzed the data separately by race, our findings of low TAC clearance remained in the white Eld patients compared with white younger recipients but did not in Eld African-origin patients. Notwithstanding, since the numbers of patients in the latter group was too small we cannot not discard the possibility that TAC clearance are significantly lower only in white Eld recipients compared with younger white Ctrls but not in African-origin Eld recipients compared with African-origin younger recipients. This possibility needs to be tested in another study with a higher number of recipients from African origin.
We have also observed a higher TAC adjusted trough-level, a higher adjusted-Cmax and a lower fluctuation among these patients. Because adjusted TAC exposure was higher in the Eld group, a higher trough level could only be the result of a higher exposure due to a decreased clearance. In the same way, a higher adjusted-Cmax could be due only to a higher concentration reached only because it started at a higher trough level. The lower fluctuation in the Eld group supports that a higher TAC bioavailability may not be the case. On the other side, we cannot exclude definitively that a diminished P-glycoprotein activity in the enterocytes is not participating in the process of an increased bioavailability as aging interferes with the expression and activity of this transporter.7
Finally, our data show that in the Eld recipient, TAC exposure can be monitored by the trough level as well as in younger recipients.30,31 In fact, the high correlation between C0 and exposure here observed emphasizes that the usual TAC trough level of 2 to 5 ng/mL, used in association with EVL, for example, gives an exposure range of 59 to 108 ng/h per mL; a wide therapeutic window that certainly needs to be redefined.
The clinical impact of our findings is that TAC should probably be started with a lower dose in Eld people to avoid early high exposure and adverse events like impaired insulin secretion.9 Another point is that a diminished TAC clearance indicates that a much lower TAC oral dose for the Eld recipient would be needed in the long run. We have experienced this problem in our geriatric transplant outpatient clinic where a minimum dose of 1 mg twice a day usually determines a higher trough level than targeted. This suggests that the use of TAC capsules of 0.5 mg, which may not be available in many countries, is necessary. These low-dose capsules will be particularly useful when very low TAC exposure is targeted in combination with mammalian target of rapamycin inhibitors.32,33
One bias of our study is the fact that not all patients performed all PK-TAC in all timepoints. However, repeated measures including patients who performed all PK studies did not achieve different conclusions.
In summary, we have shown that Eld recipients have a lower TAC clearance than younger subjects what leads to a higher TAC exposure. We have also shown that TAC trough level is a good way to monitor TAC exposure in this cohort. Elderly patients need a low-dose TAC capsules to facilitate daily dose adjustments.
Elias David-Neto is a researcher of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), Brazil.
The authors are very grateful to Professor Teun van Gelder from the Department of Internal Medicine Hospital Pharmacy, Erasmus MC, Rotterdam, Netherlands, for the extensive deep review of this article.
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