Beneficial effects of traditional Chinese medicine Wuzhi Capsule on tacrolimus dose requirements in liver transplant patients with different donor-recipient CYP3A5 genotypes


 Background/Aims: Tacrolimus (Tac) is an immunosuppressant that is widely used to prevent allograft rejection in patients after liver transplantation. Recently, a Chinese herbal medicine known as Wuzhi Capsule (WZC) was shown to increase Tac blood concentrations by inhibiting the activity of CYP3A5 in animal studies in rats. To date, it remains unexplored whether WZC can be used to reduce the dose requirement for Tac in liver transplant patients with different donor-recipient CYP3A5 genotypes. Materials and Methods: A total of 185 liver transplant patients were enrolled and were divided into four groups according to the combinations of donor-recipient CYP3A5 phenotypes. WZC was given to patients who had C0/D of Tac ≤ 1 ng/ml per mg and required a dose of Tac ≥ 4 mg. Results: The R+/D+ group had the lowest C0, C0/D, and C0/D/W among the four groups. Furthermore, a larger proportion of patients in the CYP3A5 expression groups required Tac dose adjustment to achieve a therapeutic effect and were given Tac with WZC. Notably, the use of WZC significantly increased the blood concentrations of Tac in the CYP3A5 expression groups. Conclusion: WZC significantly increased the C0, C0/D, and C0/D/W in the CYP3A5 expression groups.


Introduction
Liver transplantation (LT) is currently the only curative treatment for patients with end-stage liver disease. After LT, patients commonly require oral immunosuppressive agents as anti-rejection therapy to prevent allograft rejection. Tacrolimus (Tac) is widely used in most immunosuppressive regimes for LT patients mainly due to its high immunosuppressive effects and low drug-related side effects (6)(7)(8). However, both rejection of the transplanted liver and Tac-related adverse effects occur after LT in a proportion of patients. This is primarily because the therapeutic margin of Tac is very narrow and its metabolic enzyme, cytochrome P450 3A5 (CYP3A5), has different genetic variants (1). These factors have a signi cant impact on the pharmacokinetics of Tac and thereby affect the required dose of Tac for LT patients. In clinical practice, the Tac dose needs to be adjusted frequently according to its blood trough concentration (C0) in LT patients to achieve a therapeutic effect on postoperative rejection and to avoid drug toxicity. A number of previous studies have shown that higher doses of Tac are associated with side effects, such as liver and kidney toxicity, tumor promotion and progression, and increased risk for diabetes (2)(3)(4)(5). Therefore, there is an urgent need to reduce the Tac dose while maintaining the therapeutic effect in patients who undergo LT.
Recently, a Chinese herbal medicine named Wuzhi Capsule (WZC) was demonstrated to increase Tac blood levels in animal studies through suppressing the enzymatic activity of CYP3A5 in rats. WZC is made from Schisandra sphenanthera, also known as Nan-Wuweizi, and is an effective treatment for alleviating liver damage with various etiological factors (e.g. hepatoxins, viral hepatitis) (9,10). It has been demonstrated that WZC inhibits the P glycoprotein-mediated e ux of Tac and CYP3A-mediated metabolism of Tac, leading to an increase in the oral bioavailability and blood concentration of the drug (11).
In recent years, WZC has been prescribed for patients after solid organ transplantation in many transplant centers in China.
However, no study on the clinical application of WZCs in LT patients with different combinations of donor-recipient CYP3A5 genotypes has been conducted. To date, it remains unexplored whether WZC can exert similar effects and can be used to reduce the dose requirements for Tac in LT patients.
Intrigued by and building upon the ndings regarding Tac in animal studies, we performed the rst human study to assess the effects of WZC on Tac blood concentrations in the early phase post LT in patients with different combinations of donorrecipient CYP3A5 genotypes. The ndings from this study may improve anti-rejection therapy for patients after LT.

Materials And Methods
Patients During the period between 2017 and 2020, More than 200 patients received liver transplants from brain-dead donors and were given a triple immunosuppressive regimen based on calcineurin inhibitors (Tac, mycophenolate mofetil, methyl ketone) in the First Hospital of Jilin University (Changchun, Jilin, China) were screened for eligibility. Of these patients, 185 were enrolled in this study. The key eligibility criteria were as follows: (1) Aspartate transferase (AST) and alanine transferase (ALT) less than 10 times the upper limit of normal (ULN) values within 2 week after surgery; and (2) Survival greater than 6 months. Patients who met the following criteria were excluded from this study: (1) Taking other drugs known to affect the dose requirements of Tac, such as diltiazem, uconazole, and rifampin; (2) Occurrence of acute rejection, abdominal hemorrhage, and severe abnormal function of the liver caused by biliary stricture within 3 months; (3) Taking other immunosuppressive agents (e.g. cyclosporin A, sirolimus).
Prior to the study, all participants provided written informed consent. The clinical study was carefully reviewed and approved by the Ethics Committee of the First Hospital of Jilin University.

Treatments and measurements of Tac blood levels
The patients began taking Tac on the second day after LT. The initial dose of Tac was 2-3 mg, the target C0 measured prior to the rst dose of Tac (t = 0) was 5-10 ng/ml, and the dose of Tac was subsequently adjusted according to C0. When the dose of Tac was ≥ 4 mg and the dose-adjusted C0 (C0/D) of Tac measured twice in succession was ≤ 1 ng/ml per mg, WZC was given, starting with a dose of 0.5 g and increasing the dose as appropriate according to the C0.
Tac blood levels were monitored and quanti ed using an automatic enzyme immunoassay analyzer (Siemens Pro-E, Germany) according to the manufacturer's instructions. Given that the area under the Tac concentration-time curve (AUC) has a good correlation with the trough level (13,14), the C0 before the rst dose of Tac can accurately re ect the metabolism of the drug. All data were analyzed using the statistical software SPSS25. For continuous data, the Kolmogorov Smirnov test was performed to determine whether the data had a normal distribution. Normally distributed data, such as age and weight, were expressed as mean ± standard deviation (x ± s). Non-normally distributed data were presented in median and quarter points; that is, the "median (quartile)" M (Q1-Q3) means was used for data such as the recipient's liver function (including: AST, ALT, bilirubin, albumin), blood test (hemoglobin), and renal function (creatinine) results. Categorical variables (e.g. gender, blood type) were expressed as frequency and the chi-square test was used for comparisons. For data with a normal distribution and homogeneity of variance, one-way analysis of variance and the Student-Newman-Keuls (SNK) test were used to perform multiple comparisons between groups. The Kruskal-Wallis test was used for continuous variables with a non-normal distribution or uneven variance, such as C0, C0/D, C0/D/W. P < 0.05 was considered statistically signi cant between groups.

Results
Baseline characteristics of liver transplant patients A total of 185 LT patients were enrolled in this study, of which 138 (74.59%) were men and 47 (25.41%) were women. The baseline demographics, clinical characteristics, and laboratory tests results are summarized in Table 1. The mean age of the patients was 52.8 ± 9.9 years. The patients had different donor-recipient CYP3A5 genotypes, according to which they were divided into four groups: R+/D + group (n = 47), R+/D-group (n = 48), R-/D + group (n = 45), and R-/D-group (n = 45). Statistical analysis revealed no signi cant differences in the baseline characteristics between the groups (P > 0.05) ( Table 1). Effects of recipient-donor CYP3A5 genotypes on Tac blood concentrations Tac blood concentrations, represented by C0, C0/D, and C0/D/W, were compared between the groups with different combinations of recipient-donor CYP3A5 genotypes and the resulting data are shown in Tables 2 and 3. On day 7, the C0 was 5.600 ng/ml in the R+/D + group, which was signi cantly lower than the corresponding values of 7.900 ng/ml in the R-/Dgroup (P < 0.001) and 7.350 ng/ml (P = 0.002) in the R+/D-group. On day 14, the C0 in the R+/D + group was signi cantly lower than that in the R-/D-group (P = 0.005).
A similar comparison was performed for C0/D between the groups. The R+/D + group had signi cantly lower C0/D than the corresponding levels in the R+/D-group (P < 0.001), the R-/D + group, and the R-/D-group (P < 0.001). On day 14, the C0/D in the R-/D-group was 1.940 ng/ml per mg, which was signi cantly higher than those in the R+/D + group (P < 0.001), the R+/D-(P = 0.003), and the R-/D + group (P < 0.001).
In terms of C0/D/W, the R+/D + group had a mean level of 63.600 ng/ml per mg/kg, which was signi cantly lower than the levels in the R+/D-group (P = 0.006), the R-/D + group (P = 0.003), and the R-/D-group (P < 0.001). On day 14, the C0/D/W in the R-/D-group was 129.000 ng/ml per mg/kg, which was signi cantly higher than that in the R+/D + group (P < 0.001), the R+/D-group (P = 0.007), and the R-/D + group (P = 0.012).

Effects of WZC on early blood concentrations of Tac in patients with different combinations of donor-recipient CYP3A5 genotypes
The effects of WZC on the early blood concentrations of Tac (e.g. C0, C0/D, and C0/D/W) were assessed in the enrolled participants with different combinations of recipient-donor CYP3A5 genotypes and the results are summarized in Tables 4 and  5. Among the 185 LT patients, 153 (82.70%) did not require WZC on day 7 and were characterized as the Non-WZC group. The 32 (17.30%) patients that required WZC on day 7 were characterized as the WZC group. Notably, 112 (60.54%) patients remained in the Non-WZC group on day 14, with 73 (39.46%) in the WZC group. The patients in the Non-WZC and WZC groups were further divided into four subgroups according to the difference in the donor-recipient CYP3A5 genotypes. The differences in C0, C0/D, and C0/D/W were compared between the groups on days 7 and 14 following LT.
On day 7, the C0 of patients who did not require WZC in the R+/D + group was 5.300 ng/ml, which was signi cantly lower than the corresponding values of 7.800 ng/ml in the R-/D-group (P < 0.001), 7.000 ng/ml in the R+/D-group (P = 0.007), and 5.600 ng/ml in the R-/D + group. There was no signi cant difference in C0 between the four groups among those who received WZC on day 7 (P = 0.212). The C0 of patients who did not require WZC on day 14 in the R+/D + group was 6.200 ng/ml, which remained signi cantly lower than the values in the R-/D-group (P = 0.009) and R-/D + group. There was no signi cant difference in the C0 between the four groups with different combinations of recipient-donor CYP3A5 genotypes among patients who were treated with WZC on day 14 (P = 0.166).
Among patients who did not require WZC on day 7, the C0/D in the R+/D + group was 0.993 ng/ml per mg, which was signi cantly lower than the corresponding values in the R+/D-group (P = 0.002), R-/D + group (P = 0.003), and R-/D-group (P < 0.001). There was no signi cant difference in the C0/D between the four groups among patients who received WZC on day 7 (P = 0.138). The C0/D in the R-/D-group of patients who did not receive WZC on day 14 was 1.940 ng/ml per mg, signi cantly higher than the values in the R+/D + group (P < 0.001), the R+/D-group (P = 0.021), and the R-/D + group (P < 0.001). The C0/D in the R-/D-group of patients who received WZC on day 14 was 1.912 ng/ml per mg, signi cantly higher than that in the R+/D + group (P = 0.013).
Among patients who did not require WZC on day 7, the C0/D/W in the R+/D + group was 63.575 ng/ml per mg/kg, which was signi cantly lower than the corresponding values of 87.111ng/ml per mg/kg (P = 0.008) in the R+/D-group, 87.500 ng/ml per mg/kg (P = 0.007) in the R-/D + group, and 113.600 ng/ml per mg/kg (P < 0.001) in the R-/D-group. There was no signi cant difference between the four groups among patients who received WZC on day 7 (P = 0.378). The C0/D/W among patients who received WZC on day 14 in the R-/D-group was 129.000, which was signi cantly higher than that in the R+/D + group 61.600 ng/ml per mg/kg (P < 0.001), as well as the corresponding values of 88.280 ng/ml per mg/kg (P = 0.036) in the R+/D-group and 77.100 ng/ml per mg/kg (P = 0.002) in the R-/D + group. There was no signi cant difference among the four groups among patients who received WZC on day 14 (P = 0.098).
Self-control analysis of effects of WZC on Tac blood concentrations To further evaluate the bene cial effects of WZC on Tac blood concentrations, the LT patients were divided into four subgroups based on whether and when WZC was orally administrated ( Table 6). 112 (60.54%) patients did not receive WZC on days 7 and 14 after LT (A), 20 (10.81%) were given WZC on both days 7 and 14 after LT (B), 41 (22.16%) received WZC only on the 14th day after LT (C), and 12 (6.49%) received WZC on the 7th day, with a higher dose on the 14th day after LT. The C0, C0/D and C0/D/W were compared between these groups.
The effects of WZC on the Tac blood concentrations (C0, C0/D, and C0/D/W) were further assessed in LT patients who received WZC on the 14th day but not on the 7th day after surgery. As shown in the upper panels in Fig. 1, the C0, C0/D, and C0/D/W were 8.000 ng/ml, 1.333 ng/ml per mg, and 94.900 ng/ml per mg/kg on the 14th day, signi cantly higher than the corresponding values of 5.000 ng/ml (P < 0.001), 0.983 ng/ml per mg (P < 0.001), and 66.867 ng/ml per mg/kg (P < 0.001) on the 7th day. It was noteworthy that a higher dose of WZC led to increased effects on the C0, C0/D, and C0/D/W, which were 10.600 ng/ml, 1.830 ng/ml per mg, and 123.817 ng/ml per mg/kg in patients who received a higher dose of WZC on the 14th day. The corresponding values were 6.600 ng/ml (P = 0.034), 1.214 ng/ml per mg (P = 0.004), and 78.524 ng/ml per mg/kg (P = 0.004) on the 7th day after LT (lower panels, Fig. 1). These data provided direct evidence that WZC signi cantly elevated the Tac blood concentrations in LT patients.

Discussion
This study assessed the effects of the traditional Chinese medicine WZC on Tac blood concentrations in 185 LT patients with different combinations of donor-recipient CYP3A5 genotypes. The major novel ndings were as follows: (1) Donor-recipient CYP3A5 genotypes signi cantly affected the Tac blood concentrations, with the lowest C0, C0/D, and C0/D/W observed in the R+/D + group on days 7 and 14 after LT; (2) Tac blood levels were signi cantly elevated by the use of WZC in LT patients in the R+/D+, R+/D-, and R-/D + groups; (3) Greater increases in the C0/D and C0/D/W were signi cantly associated with higher doses of WZC; (4) The use of WZC reduced the need for Tac dose adjustment, especially in the R+/D+, R+/D-, and R-/D + groups.
Tac is a powerful immunosuppressant that is widely used for patients who undergo solid organ transplantation (8). However, the narrow therapeutic window of Tac presents a clinical challenge. For instance, too low a dose can lead to rejection, and too high a dose can cause drug-induced toxicity (15). Therefore, the target C0 is generally set at 5-10 ng/ml (16) and the oral dose of Tac is adjusted according to the blood concentration. Tac is mainly metabolized by CYP3A4 and CYP3A5 in the liver and small intestine, while P glycoprotein, a product of the multidrug resistance 1 gene (MDR1) or the so called ATP-binding cassette (ABC) transporter, ABCB1, limits the absorption of Tac by pumping it out of the cell (16,17). Due to the low frequency of polymorphisms, it is di cult to attribute large pharmacokinetic changes to the CYP3A4 genotype alone (18), and no signi cant difference between the high-and low-CYP3A4 groups in Tac dosage or the C/D ratio has been reported (16).
Although intra-individual variations in the C0/D ratio are closely related to the expression level of MDR1, there is no signi cant relationship between the C3435T single nucleotide polymorphism (SNP) in the MDR1 gene and the pharmacokinetics of Tac (16) and the impact of the SNP is rather limited (19,20).
Previous studies have demonstrated that the variations of the CYP3A5 genotypes largely contribute to Tac disposition (20)(21)(22). The SNP in intron 3 of the CYP3A5 gene (CYP3A5*3, g.6986A > G, rs776746) can cause splicing errors and abnormal splicing of mRNA to stop codons prematurely, thereby deactivating enzymes (1). Many previous studies have reported the signi cant impact of CYP3A5*3 SNPs on the pharmacokinetics of Tac, causing the enzyme to lose its activity (16,23,24). In CYP3A5 expressers, the weight-corrected Tac daily dose requirements and weight-corrected steady-state clearance were ~ 1.8fold higher, while the dose-corrected C0 and AUC0-12 were ~ 2.0-fold lower than those of CYP3A5 non-expressers (25). Intestinal CYP3A5 and hepatic CYP3A5 play important roles in the oral clearance of Tac (16). In this study, according to the donor-recipient CYP3A5 genotypes, the185 LT recipients were divided into four groups: R+/D + group, R+/D-group, R-/D + group, R-/D-group. The different combinations represented the intestinal and hepatic CYP3A5 activity. We found that the C0/D and C0/D/W in the R+/D + group were the lowest among the groups on the 7th day, while the C0/D and C0/D/W of the R-/Dgroup were signi cantly higher than those of the other three groups on the 14th day. These ndings are consistent with previous studies (16,24), indicating that either the intestinal enzyme activity or the liver enzyme activity improved on day 14 after LT. Unlike the intestinal CYP3A5 enzyme, which is responsible for stronger Tac metabolism than the liver CYP3A5 enzyme in the early stages after LDLT as reported previously (11,26), this study has shown that the C0, C0/D, and C0/D/W of the R+/Dand R-/D + groups are not signi cantly different on days 7 and 14 after LT. This indicates that the CYP3A5 enzymes in the intestine and liver have no signi cant differences in Tac metabolism in the early stages after LT.
WZC is a traditional Chinese medicine extracted from Schisandra sphenanthera and is widely used to treat liver damage caused by hepatoxins, viral hepatitis, or acetaminophen (9,10,27). Its active ingredients are schisandrins (A, B, C), schisandrols (A, B), and schisantherins (A, B) (28). Qin et al reported that the lignans in WZC and Tac were both substrates of CYP3A, the a nity of the WZC lignans to CYP3A was much higher than that of Tac, and WZCs successfully competed against Tac with a much stronger metabolism (29). Studies in rats and healthy volunteers have demonstrated that the active ingredients of WZC may increase the oral bioavailability of Tac and maintain its blood concentration by inhibiting the metabolism of CYP3A-mediated Tac and the P glycoprotein-mediated e ux of Tac (11,14). Co-administration of Wuzhi tablet signi cantly reduced the Tac dose requirements without impairing its immunosuppressive effect (30). In the present study, we explored the potentially bene cial effects of WZC in patients with different donor-recipient CYP3A5 genotypes. On day 7 after LT, the C0/D and C0/D/W in the R+/D + group were signi cantly lower than the values in the other three groups. On day 14, the C0/D and C0/D/W of the R-/D-group was signi cantly higher than those of the other three groups. It may merit attention that the use of WZC diminished the difference in Tac metabolism caused by variations in the CYP3A5 genotypes, thereby reducing the required dose of Tac in the enzyme expression groups (R+/D+, R+/D-, and R-/D + groups).
Qin and colleagues reported that the AUC value after oral Tac dosing increased by 2.1 fold and that the oral bioavailability (Foral) of Tac increased from 5.4-13.2% when co-administered with WZC (11). In this study, we observed that WZC signi cantly increased the C0, C0/D, and C0/D/W on day 14 compared with the values on day 7. It can be concluded that WZC can signi cantly increase the blood concentration of Tac and has a superimposing effect; that is, increasing the dose of WZC can further increase the blood concentration of Tac. No organs from executed prisoners were used in our study. The study protocal was approved by the ethical committee of Bethune Hospital 1, China. Written consent for publication of the article was obtained.

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All authors approved the nal version for publication.

Availability of data and materials
We are committed to the reliability of the data used

Competing interests
All authors declare there were no con ict of interests involved.       Table 5 Pairwise comparison between groups  Tac concentrations  P1  P2  P3  P4  P5  P6  C0  7th