Effect of Azole/Echinocandin Use on Tacrolimus Pharmacokinetics in Kidney Transplant Recipients
Conditions: Kidney Transplant Patients
Sponsor: LI YAN

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Research Program 
(Version number: V2.0 Version date: 2023.01.28) 
 
 
 
 
Project Title: Effect of Azole/Echinocandin Use on Tacrolimus 
Pharmacokinetics in Kidney Transplant Recipients 
Project origin: Investigator-initiated 
Department:  
Principal Investigator:  
 
 
Investigator statement and protocol signature page 
 
As the main person in charge of this research project, I will follow the ethical 
principles of the Ministry of Health's Measures for Ethical Review of Biomedical 
Research Involving Human Beings (2016), the WMA Declaration of Helsinki (2013) 
and the CIOMS International Ethical Guidelines for Biomedical Research on Human 
Beings (2002), and the GCP, and will conduct the study according to the requirements 
of the present protocol under the guidance of the Code of Practice for Quality 
Management of Pharmaceutical Clinical Trials using the protocols approved by the 
Ethics Committee in order to ensure the scientific validity of the study and to protect 
the health and rights of the subjects. 
 
 
 
Name： 
Date：January 28, 2023 

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Program Summary 
Program Title 
Effect of Azole/Echinocandin Use on Tacrolimus 
Pharmacokinetics in Kidney Transplant Recipients 
Version No./Version 
Date 
V2.0/2023.01.28 
Principal Investigator 
 
Nature of Research 
A retrospective cohort study 
Study Objective 
To investigate the effect of azoles/echinocandins on 
tacrolimus C0, D and C0/D 
Sample Size 
Approximately 521 inpatients using target medications from 
January 1, 2015 to April 1, 2023 to be included 
Research Population 
Inpatients on target medications from January 1, 2015 to 
April 1, 2023 
Research Method 
Non-interventional, retrospective methods of data analysis 
Inclusion Criteria 
1. First time kidney transplantation; 
2. Taking a tacrolimus-based triple immunosuppressive 
regimen (tacrolimus + sodium mescaline enteric tablets + 
glucocorticoids) after renal transplantation; 
3. Age ≥ 18 years. 
Exclusion criteria 
1. Combined multi-organ transplants; 
2. Pregnant and lactating women; 
3. Severe impairment of liver function or severe 
gastrointestinal disease, gastrointestinal resection, 
malabsorption syndrome; 
4. Allergy or intolerance to macrolides, mycophenolic acid 
or glucocorticoids; 
5. Missing or incomplete clinical information and 
postoperative follow-up data (e.g., missing target drug 
concentration, dose, etc.); 
6. Recipients who had their grafts removed or died due to 
rejection or serious complications within 1~2 months after 
surgery; 
7. Poor compliance and accuracy of results (e.g. irregularity 
in blood collection time); 
8. Postoperative co-application of other drugs that affect the 
blood concentration of target drugs such as tacrolimus or 
voriconazole or caspofungin (e.g. pentothal capsules, 
rifampicin, phenytoin sodium, carbamazepine, etc.) at the 
same time. 
Key Efficacy 
Indicators 
The C0/D of tacrolimus 
Secondary Efficacy 
Indicators 
The C0 and D of tacrolimus 

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Security Indicators 
Acute rejection, delayed recovery of transplanted kidney 
function, death 30 days after surgery, postoperative 
infection 
Statistical Analysis 
Method 
chi-square test, independent samples t-test, paired samples t-
test, two-sample Wilcoxon rank sum test, Mann-Whitney U 
test 
Forms of publication 
of research results 
academic paper 
 
1. Objective 
This 
study 
investigated 
the 
effect 
of 
combined 
antifungal 
drugs 
(voriconazole/posaconazole/caspofungin/micafungin) on the trough concentration of 
tacrolimus in renal transplant recipients and analyzed the effects of genetic factors 
(CYP3A5, CYP2C19) and clinical factors on the drug-drug interactions between 
antifungal drugs (voriconazole/posaconazole/caspofungin/micafungin) and tacrolimus 
with a view to providing a reference basis for the rational use of tacrolimus and 
antifungal drugs in the rational use of clinical medication to provide a reference basis. 
2. Background 
Chronic kidney disease (CKD) has become a global public health problem. 
According to statistics, the prevalence of CKD is 11%~13% worldwide, and about 700 
million people suffer from CKD in varying degrees [1]. 2022 Meta-analysis showed that 
the prevalence of CKD among Chinese adults is 13.1%. With the progression of CKD, 
the function and structure of the kidney are damaged, manifested by renal hypoplasia, 
and even renal failure in the end stage [2], and all organs of the patient's body may be 
involved, which not only causes great physical and mental damage, but also leads to 
the loss of personal and social medical costs. Kidney transplantation is the most 
effective clinical treatment for patients with end-stage renal failure [3], providing better 
survival and quality of life compared with long-term dialysis [4]. 
For renal transplant recipients, immunosuppressive drugs should be administered 
for a long time after surgery to prevent auto rejection, and the choice of 
immunosuppression is often based on the calcineurin inhibitor (CNI) as a two-
combination (CNI + mycophenolate mofetil/glucocorticoid) or three-combination (CNI 

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+ mycophenolate mofetil + glucocorticoid) immunosuppressive regimen. Since renal 
transplant patients need to receive immunosuppressive therapy for a long period of time, 
how to apply immunosuppressive drugs safely and effectively is the key to whether the 
transplanted kidneys can maintain a long enough survival time and whether the patients 
can achieve a high quality of life. Tacrolimus (TAC) and cyclosporin are the calcineurin 
inhibitor (CNI) [5], and compared with cyclosporin, TAC can reduce the incidence of 
rejection (42.1% vs. 18.4%, P=0.03) [6] and improve the graft survival. TAC can reduce 
the incidence of rejection (42.1% vs. 18.4%, P=0.03) [6], and increase the survival rate 
of grafts [7], and has been gradually replacing cyclosporine as the first-line 
immunosuppressant because of its significant therapeutic efficacy and low adverse 
effects. 
These immunocompromised patients are more susceptible to infections due to 
prolonged immune system suppression, which is currently the leading cause of death in 
solid organ transplantation (SOT) recipients. Currently, infection is the leading cause 
of death in solid organ transplantation (SOT) recipients, and invasive fungal disease 
(IFD) remains the leading cause of morbidity and mortality in solid organ transplant 
recipients after SOT. Epidemiologic surveys abroad have shown that the pathogens of 
postoperative IFD in SOT recipients are mainly Candida (53.0%-59.0%) and 
Aspergillus (19.0%-24.8%). In clinical treatment, the commonly used antifungal agents 
are triazoles and echinocandins. 
Most antifungal drugs in vivo are metabolized by cytochrome P450 (CYP450) 
enzymes in the liver, e.g., voriconazole is metabolized by CYP2C19, CYP2C9, and 
CYP3A4 enzymes, and micafungin is metabolized by CYP1A2, CYP2B6, CYP2C, and 
CYP3A. In contrast, SOT recipients require long-term use of TAC, which is 
metabolized mainly by CYP450 3A enzymes in the liver and small intestine, with the 
CYP3A4 and CYP3A5 isoenzymes contributing significantly [8]. Studies have shown 
that there are drug-drug interactions between antifungal drugs and TAC, which can 
cause changes in TAC blood levels. Co-administration of caspofungin reduced the peak 
concentration (Cmax) of TAC by approximately 20% [9]. Another study [10] found that 
VRC affects TAC metabolism mainly related to polymorphisms in the CYP2C19 gene. 

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Guidelines suggest that when VRC is initiated in SOT recipients already receiving TAC, 
the dose of tacrolimus should be reduced to one-third of the usual dose. However, drug-
drug interactions between VRC and TAC have significant inter-individual variability 
among recipients, and empirically based dose adjustments can still result in significant 
TAC overdose [11], so dose adjustments for TAC at the time of initiating or discontinuing 
VRC are not uniform. 
There is a drug-drug interaction between VRC and TAC that leads to an increase 
in TAC trough concentrations; however, there is significant inter-individual variability 
in this effect, and a reduction in the dose of TAC to one-third of the usual dose of VRC 
in combination with TAC, as recommended by the guideline, is not sufficient to meet 
the clinical need [11]. Studies of VRC in combination with TAC have focused on bone 
marrow and lung transplant recipients, with fewer studies in renal transplant recipients 
[11-13]. Caspofungin and micafungin, both of which are metabolized by the liver, may 
have drug-drug interactions with TAC, which may affect the TAC trough concentration, 
whereas there are fewer reports of drug-drug interactions between micafungin and TAC. 
Base on the previous studies, this study was conducted to investigate the effects of 
CYP3A5 and CYP2C19 gene polymorphisms on the trough concentration of TAC after 
renal transplantation, to investigate the effects of antifungals on the trough 
concentration of TAC, and to analyze the effects of genetic factors on drug interactions 
between antifungals and TAC, in order to provide a reference basis for rational use of 
TAC and antifungals in clinical practice. The effects of genetic factors on the interaction 
between antifungal drugs and TAC were also analyzed, with a view to providing a 
reference basis for the rational use of TAC and antifungal drugs in clinical practice. It 
is of great significance to prevent and control fungal infections after renal 
transplantation and at the same time make TAC reach the therapeutic target 
concentration as early as possible, in order to minimize the occurrence of immune 
rejection after renal transplantation, to reduce the adverse reactions and toxic effects of 
TAC, to improve the safety and effectiveness of TAC, and to achieve the optimal 
therapeutic effect of TAC. 
References: 

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[1] Hill N R, Fatoba S T, Oke J L, et al. Global Prevalence of Chronic Kidney Disease - A Systematic 
Review and Meta-Analysis[J]. PLoS One, 2016, 11(7): e0158765. 
[2] Webster AC, Nagler EV, Morton RL, et al. Chronic Kidney Disease[J]. Lancet (London, 
England), 2017, 389(10075): 1238-1252. 
[3] Li CJ, Li L. Tacrolimus in preventing transplant rejection in Chinese patients--optimizing use[J]. 
Drug Des Devel Ther, 2015, 9: 473-485.  
[4] Yanik MV, Seifert ME, Locke JE, et al. CYP3A5 genotype affects time to therapeutic tacrolimus 
level in pediatric kidney transplant recipients[J]. Pediatr Transplant, 2019, 23(5): e13494.  
[5] Zhu W, Xue L, Peng H, et al. Tacrolimus population pharmacokinetic models according to 
CYP3A5/CYP3A4/POR genotypes in Chinese Han renal transplant patients[J]. Pharmacogenomics, 
2018, 19(13): 1013-1025.  
[6] Cheung CY, Chan HW, Liu YL, et al. Long-term graft function with tacrolimus and cyclosporine 
in renal transplantation: paired kidney analysis[J]. Nephrology (Carlton, Vic.), 2009, 14(8): 758-
763.  
[7] Dashti-Khavidaki S, Ghaffari S, Gohari M, et al. Tacrolimus Dose Requirement in Iranian 
Kidney Transplant Recipients within the First Three Weeks after Transplantation[J]. International 
journal of organ transplantation medicine, 2016, 7(3): 167-171.  
[8] Staatz CE, Goodman LK, Tett SE. Effect of CYP3A and ABCB1 single nucleotide 
polymorphisms on the pharmacokinetics and pharmacodynamics of calcineurin inhibitors: Part I[J]. 
Clin Pharmacokinet. 2010, 49(3): 141-175 
[9] Yeoh S F, Lee T J, Chew K L, et al. Echinocandins for management of invasive candidiasis in 
patients with liver disease and liver transplantation[J]. Infect Drug Resist, 2018, 11: 805-819. 
[10] MORIYAMA B, OBENG AO, RARBARINO J, et al. Clinical Pharmacogenetics 
Implementation Consortium (CPIC) Guidelines for CYP2C19 and Voriconazole Therapy. [J].Clin 
Pharmacol Ther, 2017, 102(1): 45-51. 
[11] Vanhove T, Bouwsma H, Hilbrands L, et al. Determinants of the Magnitude of Interaction 
Between Tacrolimus and Voriconazole/Posaconazole in Solid Organ Recipients[J]. Am J Transplant. 
2017, 17(9): 2372-2380. 
[12] Cheng S, Tang M, Du J, et al. Effects of antifungal drugs on the plasma concentrations and 
dosage of tacrolimus in kidney transplant patients[J]. Eur J Hosp Pharm, 2022, 29(4): 202-206. 
[13] Kieu V, Jhangiani K, Dadwal S, et al. Effect of isavuconazole on tacrolimus and sirolimus 
serum concentrations in allogeneic hematopoietic stem cell transplant patients: A drug-drug 
interaction study[J]. Transpl Infect Dis, 2019, 21(1): e13007. 
3. Content of the Study 
3.1 Trial population 
All hospitalized patients who meet the inclusion criteria from January 1, 2015 to 
April 1, 2023 will be included in this study as the study population, and the data will 
be obtained from the Lianzhong Digital Case Browser, LIS Result View and HIS system 
of the First Affiliated Hospital of Shandong First Medical University. 
3.2 Quality control measures 
 

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Quality control was carried out throughout the study. 
(1) Collecting data during the study time period to ensure that the entered data are 
consistent with the original data. 
(2) Verify the data and organize the data. 
(3) Data analysis and verification by another person. 
3.3 Sample size calculation 
In the part of the study on the interaction of tacrolimus with triazoles, tacrolimus 
C0/D was used as an index of outcome based on the results of the pre-test of the clinical 
data of the patients with co-administration of voriconazole/posaconazole. Tacrolimus 
C0/D was 753.11±562.35 ng/ml/(mg/kg/d) in patients in the group with co-administered 
voriconazole and 143.26±99.78 ng/ml/(mg/kg/d) in the group without co-administered 
triazoles. To obtain 90% statistical validity, a two-sided alpha value of 0.05 was set to 
detect differences between groups. The total sample size for this study was calculated 
using the Performance Analysis and Sample Size (PASS) software (version 15.0), and 
a t-test was selected to obtain a sample size of at least 12 cases in each of the combined 
voriconazole group and the non-combination group. Tacrolimus C0/D was 
540.51±347.88 ng/ml/(mg/kg/d) in patients in the co-voriconazole group and 
tacrolimus C0/D was 143.26±99.78 ng/ml/(mg/kg/d) in the non-combination group. To 
obtain 80% statistical validity, a two-sided alpha value of 0.05 was set to detect 
differences between groups. The total sample size for this study was calculated using 
the Performance Analysis and Sample Size (PASS) software (version 15.0), and a t-test 
was selected to obtain a sample size of at least 9 cases in each of the combined 
posaconazole group and the non-combination group. 
In the part of the study on the interaction between tacrolimus and echinocandins, 
recipients who underwent kidney transplantation at the First Affiliated Hospital of 
Shandong First Medical University (Qianfoshan Hospital, Shandong Province, China) 
and were taking the antifungal medication caspofungin/micafungin for tacrolimus 
blood concentration monitoring after the operation between January 1, 2015, and April 
1, 2023, were selected, and tacrolimus combined with caspofungin medication group 
was collected from 171 recipients, 24 recipients in the tacrolimus combined with 

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micafungin drug group, and 305 recipients in the no antifungal drug group. In summary, 
at least 521 kidney transplant recipients are needed. 
3.4 Treatment of missing values 
Missing data or illogical data are handled by deleting cases containing missing 
values or by using missing value filling methods (mean value interpolation, etc.). The 
appropriate processing method is selected later on a case-by-case basis.  
3.5 Specific research content 
To study the effect of co-administration of azoles/echinocandins on tacrolimus C0, 
D and C0/D; before and after co-administration of tacrolimus C0/D according to 
CYP3A5 genotype stratification analysis. To study the effect of CYP2C19 and 
CYP3A5 genotypes on tacrolimus in combination with voriconazole. 
4. Method 
4.1 Enrollment criteria 
4.1.1 Inclusion criteria 
(1) First time kidney transplantation; 
(2) Taking a tacrolimus-based triple immunosuppressive regimen (tacrolimus + 
sodium mescaline enteric tablets + glucocorticoids) after renal transplantation; 
(3) Age ≥ 18 years 
4.1.2 Exclusion criteria 
(1) Combined multi-organ transplants; 
(2) Pregnant and lactating women; 
(3) Severe impairment of liver function or severe gastrointestinal disease, 
gastrointestinal resection, malabsorption syndrome; 
(4) Allergy or intolerance to macrolides, mycophenolic acid or glucocorticoids; 
(5) Missing or incomplete clinical information and postoperative follow-up data (e.g., 
missing target drug concentration, dose, etc.); 
(6) Recipients who had their grafts removed or died due to rejection or serious 
complications within 1~2 months after surgery; 
(7) Poor compliance and accuracy of results (e.g. irregularity in blood collection 
time); 

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(8) Postoperative co-application of other drugs that affect the blood concentration of 
target drugs such as tacrolimus or voriconazole or caspofungin (e.g. pentothal 
capsules, rifampicin, phenytoin sodium, carbamazepine, etc.) at the same time. 
4.2 Data collection 
Kidney transplant patients were strictly screened according to the NaCl criteria, 
and the patients' basic information (name, hospitalization number, contact information, 
gender, age, height, weight), medical history (diabetes mellitus and hypertension), the 
time of kidney transplantation, tacrolimus medication (time of medication and daily 
dosage), and its trough concentration (C0), co-medication (time of medication and daily 
dosage, and the types of co-medications including posaconazole, voriconazole, 
caspofungin and micafungin), and the patients' liver and biliary function indexes, 
including aspartate aminotransferase (AST), alanine aminotransferase (ALT), 
glutamyltransferase (GGT), alkaline phosphatase (ALP), total bilirubin (TBIL), and 
direct bilirubin (DBIL), etc., and the calculation of tacrolimus trough blood 
concentration/daily dose ratio (C0/D ) [(ng/ml/)/(mg/kg/d)]. 
4.3 Methods of measuring the study indicators 
The required C0 and D values for tacrolimus were filtered from the United Digital 
Case Browser, and the tacrolimus C0/D values were entered and calculated in Excel. 
4.4 Data management method 
All data were uniformly entered and saved in Excel, statistically analyzed in SPSS 
22.0, and the results of the analysis were also uniformly presented by Excel, and plots 
were made with GraphPad Prism 8.0. 
5. Trial Procedures 
5.1 Subject management 
1) Recruitment method of subjects 
This is a retrospective study with data from Lianzhong Digital Case Browser, LIS 
Result View and HIS system of the First Affiliated Hospital of Shandong First Medical 
University, free of subject recruitment. 
2) Informed consent process 
As this study was a retrospective study, the informed consent process was exempted. 

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6. Start and End of the Trial 
The time period of the extracted EHR data is from January 1, 2015 to April 1, 2023; 
The study period for this project is from February 1, 2023 to June 1, 2024. 
7. Ethical Principles and Compliance with Relevant Regulations 
The study strictly followed the current Declaration of Helsinki (2013) as well as 
relevant laws and regulations applicable within the People's Republic of China and the 
review opinions of the Ethics Committee. 
8. Statistical Analysis 
SPSS 22.0 software was used to statistically analyze the data; propensity score 
matching was taken to match the baseline data of kidney transplant patients in the 
combination and non-combination groups, and a new dataset with successful matching 
was obtained, and normality test was performed by Shapiro-Wilk method. Measures 
conforming to normal distribution were expressed as (mean±sd), non-normal measures 
as median (interquartile spacing), and counts as frequency (rate). Between groups: 
independent samples t-test was used for measures that met normal distribution, Mann-
Whitney U test was used for measures that did not meet normal distribution, and chi-
square test was used for count data to analyze the effects of genetic polymorphisms, 
azoles/echinocandins on the trough concentration of tacrolimus in renal transplantation 
recipients; within groups: paired samples t-test was used for measures that met normal 
distribution, Wilson's t-test was used for measures that did not meet normal distribution, 
and Wilson's t-test was used for measures that were not distributed. Wilcoxon test was 
used to analyze the changes in tacrolimus trough concentration in renal transplant 
recipients before and after co-administration of azoles or echinocandins. p<0.05 
indicates that the difference is statistically significant. The chi-square test was used to 
analyze whether the genotype distribution conformed to Hardy-Weinberg equilibrium, 
and P＞0.05 indicated that it conformed to population genetic balance. 
9. Form of Publication of Research Results 
The research results will be published in the form of academic papers.