Effects of Triazole Antifungal Agents on the Plasma Concentration and Dosage of Cyclosporin in Patients with Aplastic Anaemia

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Introduction
Aplastic anaemia (AA) is a bone marrow haematopoietic failure syndrome.Its annual incidence is 0.74/100,000 in China, and it can occur in all age groups [1].Serious aplastic anaemia can cause fatalities, with a mortality rate of 75% [2].Te pathogenesis of AA is unclear; however, T-lymphocytemediated immune damage to haematopoietic stem cells is the pathological basis of its development, and abnormally high expression of immune molecules plays a vital role in the pathogenesis of AA.Terefore, immunosuppressive drugs have become the frst-line treatment for patients with AA [3].Cyclosporine (CsA) has been recommended as the treatment of choice for immunosuppressants in patients with AA [1].
Patients with haematological diseases are susceptible to various infections because of their low cellular immunity, especially during immunosuppressive therapy, which further suppresses the immune system and makes them more susceptible to invasive fungal infections (IFIs) [4].IFIs have become an important cause of morbidity and mortality in patients undergoing immunosuppressive therapy or haematopoietic stem cell transplantation [5].According to the Diagnostic Criteria and Terapeutic Principles for Invasive Fungal Disease in Patients with Haematological Diseases/ Malignancies (6th Revision), Candida and Aspergillus are the common causative agents of IFIs in patients with haematological disorders [6], and triazole antifungals are commonly used for the prevention or treatment of invasive fungal infections [5][6][7][8].Terefore, triazole antifungals are often co-administered with CsA in the treatment of patients with AA.
However, triazole antifungals drugs and CsA carry the risk of drug-drug interactions because of their efects on cytochrome P450 (CYP) enzymes and transporter proteins.CsA are substrates of CYP3A4 and p-glycoprotein (P-gp).It interacts with various drugs, including antibiotics, antifungals, and glucocorticoids [9].Posaconazole (POS), fuconazole (FCZ), and voriconazole (VCZ) inhibit CYP3A4 enzyme activity to varying degrees.POS is both an inhibitor of CYP3A4 and a substrate for P-gp, which afects the blood concentration of CsA via two pathways.POS and VCZ are potent inhibitors of CYP3A4 activity, whereas FCZ is a moderate inhibitor of CYP3A4 [10][11][12][13][14][15].It can reduce the metabolism of CsA by inhibiting the activity of hepatic enzymes, resulting in an increase in the blood concentration of CsA, which is prone to cumulative poisoning.
Although the concomitant use of azoles and CsA can result in interactions [16][17][18], most studies have focused on the interaction between CsA and triazole antifungal drugs in patients undergoing haematopoietic stem cell transplantation (HSCT) [19][20][21][22][23][24][25].Tere are limited studies on the co-administration of CsA with triazole antifungal agents in patients with AA.HSCT is one of the treatments for AA.AA is a heterogeneous bone marrow failure syndrome and that manifests mainly as red cell aplasia and reduced peripheral pancytopenia numbers.However, 50% to 60% of CsA in whole blood is distributed in red blood cells, and the pathological state of patients with AA will afect the distribution of CsA in the blood, which may be diferent from patients with HSCT.Tere are limited research studies on interaction between CsA and azoles in patients with AA.In this study, the efects of triazoles on CsA blood levels in patients with AA were explored.We also compared the efects of diferent triazole antifungal agents on CsA blood concentration and dosage in patients with AA to provide a basis for developing individualised regimens for administering CsA and triazole antifungal drugs.

Study Design and Subjects.
Tis study was conducted at the Second Hospital of the Shanxi Medical University between January 2018 and December 2022.Electronic medical records were used to identify patients with AA, severe AA, or very severe AA, and were investigated to identify patients who were treated with CsA soft capsule or CsA injection, coadministered with triazole antifungal agents (e.g., posaconazole oral suspension, fuconazole capsules or fuconazole tablets, voriconazole capsules or voriconazole tablets) to prevent or treat fungal diseases.Each patient's blood concentration of CsA was measured at least once when CsA was monotherapy and at least once when coadministered with triazole antifungal drugs.Te exclusion criteria were as follows: (i) use of other drugs that afect CYP3A4 or P-gp during hospitalisation, such as macrolide antibiotics, rifampin, carbamazepine, phenobarbital, phenytoin sodium, and HIV protease inhibitors (e.g., ritonavir and nelfnavir); (ii) age <18 years; (iii) detailed data not available; (iv) immunosuppressive therapy with CsA <3 days; and (v) liver or kidney dysfunction.Hepatic impairment was defned as alanine aminotransferase (ALT), aspartate aminotransferase (AST), or alkaline phosphatase (ALP) ≥ fve times the upper limit and total bilirubin (TBIL) ≥ two times the upper limit.Renal dysfunction was defned as a serum creatinine level >2.0 mg/dL or glomerular fltration rate <30 mL/min.
Tis study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University.Te requirement for informed consent was waived as only retrospective data were collected.

Data Collection.
Related clinical data of patients were collected.General biological data of the patients included sex, age, height, and weight.Te patients' medication information included the dosage of CsA, dosage form, time of triazole antifungal drug co-administration, and other concomitant medications.Te biochemical indicators included ALT, AST, TBIL, total protein (TP), creatinine, urea, white blood cells, red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT), and platelets (PLT).Steady-state trough concentrations were collected for all patients treated with CsA alone and co-administered with triazole antifungal drugs.Data collected for CsA monotherapy included data before the coadministration of a triazole antifungal agent or three days after the discontinuation of the triazole antifungal agent.

Terapeutic Drug Monitoring.
Whole-blood trough CsA concentrations were measured by an enzyme multiplied immunoassay technique (EMIT 2000 TDM; Syva Viva-E drug concentration analyser).Te target blood concentration of CsA for AA in adults (C 0 trough concentration) was 150-250 ng/mL [1].

Statistical Method.
All continuous variables were described as mean ± SD or median (interquartile range) based on whether they ftted the normal distribution.Normality was tested using the Shapiro-Wilk test.Statistically significant diferences in the concentration/dose (C/D) ratio with and without azole co-medication were assessed using a paired t test if the data conformed to a normal distribution; otherwise, the Wilcoxon rank test was applied.Qualitative data are presented as rates and were compared by Fisher's exact test.All analyses were performed using the IBM SPSS Statistics version 25 (IBM, New York, NY, USA).Statistical signifcance was set at P < 0.05.

Patient Demographics.
From January 2018 to December 2022, 5775 CsA concentration samples were monitored at the Second Hospital of Shanxi Medical University.Based on the inclusion and exclusion criteria, 27 hospitalisation records of 25 patients (74 samples of CsA data) were included in this study, as shown in Figure 1.Of the 25 patients, two patients had two hospitalisation records, including one patient who was co-administered with POS and FCZ during two hospitalizations, respectively.Te other patient was coadministered with FCZ and VCZ, respectively.Te records of 27 hospitalizations were stratifed into FCZ (n � 18), POS (n � 7), and VCZ (n � 2) groups based on the diferent triazole agents.In the FCZ group, there were 18 samples analysed during CsA monotherapy, while 33 samples were analysed during co-administration with FCZ.In the POS group, 7 samples during CsA monotherapy and 11 samples from co-administration with POS were analysed.In the VCZ group, two samples were analysed during CsA monotherapy, and three samples were analysed after co-administration with VCZ.Owing to the small amount of data in the VCZ group, the analysis was restricted to descriptive methods, and no statistical analyses were performed.Detailed information on the patients' characteristics is presented in Table 1.Tere were no signifcant diferences between the FCZ and POS groups in terms of basic demographic data and clinical examination fndings at the time of initial admission (P > 0.05).

Changes in CsA C/D Ratio with Triazole Antifungal Agents
Co-Terapy.Te concentration/dose (C/D) ratio was evaluated in this study because the dose of CsA was changed in some patients during the study period.To eliminate the efect of the dose on the concentration, conduct a dose correction for the CsA concentration.We compared the change in the mean C/D ratio of CsA between CsA monotherapy and co-administration with triazole antifungal drugs (Figure 2, Table 2).In the FCZ group, the mean C/D ratio of CsA montherapy was 0.54 ± 0.34 (ng/mL)/(mg/day).After co-administration with FCZ, the mean C/D ratio of CsA was 0.95 ± 0.48 (ng/mL)/(mg/day), signifcantly higher than the ratio of CsA montherapy (P < 0.05).Te mean C/D ratio of CsA with FCZ co-medication increased by 1.76 times.In the POS group, the mean C/D ratio of CsA increased from CsA monotherapy 0.75 ± 0.25 (ng/mL)/(mg/ day) to 1.48 ± 0.73 (ng/mL)/(mg/day) when co-administered with POS (P < 0.05).Te mean C/D ratio of CsA with POS co-medication increased by 1.97 times.Te change in the C/D ratio of CsA after co-administration with azoles increased signifcantly as shown in Figure 2. Tere were statistically signifcant diferences in the CsA C/D ratio between CsA monotherapy and co-administration with triazole antifungal agents (P < 0.05).
Te individual diferences of the patients in the CsA C/D ratio were analysed (Figure 3).Compared to the C/D ratio of CsA monotherapy, 88.9% (16/18) of patients had an increased C/D ratio with FCZ co-medication, and the median increase in CsA C/D ratio in the FCZ group was 0.42 (0.04-1.06) (ng/mL)/(mg/day).In addition, 85.7% (6/7) of the patients had an increased C/D ratio with POS comedication, and the median increased C/D ratio of CsA with POS co-medication was 0.63 (0.076-1.84) (ng/mL)/ (mg/day) in the POS group.Tere was a signifcant difference in the CsA C/D ratio when co-administered with FCZ or POS (P < 0.05).

Changes in CsA C/D Ratio Over Time after Triazole
Antifungal Agents Co-Terapy.Tese changes were further investigated to determine the infuence of the triazole antifungal agent co-administration time on C/D ratio.Based on the co-administration with triazole time, the two groups of patients were also divided into two subgroups: comedication (1-7 d) and co-medication (8-21 d), respectively (Table 2).During the co-administration with FCZ (1-7 d) and (8-21 d), the mean C/D ratios of CsA were 0.77 ± 0.37 (ng/mL)/(mg/d) and 0.95 ± 0.54 (ng/mL)/(mg/ d), respectively.Te mean C/D ratio with FCZ comedication (1-7 d) was approximately 1.43 times greater than CsA alone, and the mean C/D ratio with FCZ comedication (8-21 d) was approximately 1.76 times greater than CsA alone.Te mean C/D ratios for co-administration with POS (1-7 d) and (8-21 d) were 1.30 ± 0.58 (ng/mL)/ (mg/d) and 1.87 ± 1.15 (ng/mL)/(mg/d), respectively.Te C/D ratio with POS co-medication (1-7 d) was approximately 1.73 times greater than CsA alone.Te C/D ratio with co-medication (8-21 d) was approximately 2.49 times greater than CsA alone.Tese results indicated that the CsA C/D ratio was signifcantly associated with the coadministration time in the FCZ and POS groups.Te C/D ratio of CsA was plotted against the number of days after co-administration with FCZ or POS.Te level of CsA increased with the time of co-administration of azoles in the groups.Tis was a signifcant diference in the CsA C/D ratio over time when co-administered with FCZ or POS (P < 0.05).

Changes in CsA Dose with Triazole Antifungal Agents Co-Administration.
Te mean daily dose of CsA decreased after triazole antifungal agent co-administration (Table 3).In the FCZ group, the dose of CsA was decreased from 3.50 ± 1.21 (mg/kg/d) to 3.39 ± 0.94 (mg/kg/day), and the mean daily dose of CsA was decreased by 0.

Efects of Co-Administration with Triazole Antifungal
Agents on Laboratory Data of Patients.Laboratory data of AA patients administered with CsA alone and when coadministered with azole antifungal agents are shown in Table 4.No abnormalities in the biochemical indices were observed.Tere was no signifcant diference in the laboratory data of patients during CsA montherapy or the coadministered with triazole antifungal agents (P > 0.05).

Discussion
It is worth investigating whether there are diferences in the degree of interaction between triazole antifungal drugs and CsA in diferent patient populations.Tis is because it impacts whether it is necessary to preemptively reduce the dosage of CsA according to the instructions when coadministered with triazole antifungal drugs in patients with diferent diseases.Hamidreza et al. [26] found that the mean C/D ratio of CsA increased 8.40%-174.10%when co-administered with VCZ in patients received HSCT.POSA increased the mean C/D ratio of CsA to a similar extent in patients with aplastic anaemia in current study (7.60%-184.00%).Studies from Xue et al. [27] and Zhu et al. [28] indicated that the CsA C/D ratio was found to be approximately 1.5-fold higher after POS initiation in patients underwent HSCT.Here, we showed that the CsA C/D ratio increased 1.97 times when co-administered with POSA in aplastic anaemia patients.It is not clear yet if there were diferences in the degree of such interactions in these two patient populations.More studies are needed to confrm this.
Furthermore, we compared the efects of diferent triazole antifungal agents on CsA blood concentration and dosage in patients with AA.We observed that POS had a greater efect on the blood concentration of CsA than FCZ.Tese fndings may be related to and the intensity of azole drugs' inhibition of hepatic drug enzymes.POS is a strong CYP3A4 inhibitor.VCZ is metabolised by CYP3A4 and is  a moderate CYP3A4 inhibitor.Because the inhibition of CYP3A4 by POS is stronger than that by FCZ, Te coadministration with POS in patients with AA may have a greater efect on the CsA than co-administration with FCZ.
Wide interindividual variability in the magnitude of the drug interactions between triazole antifungal drugs and CsA was observed in our study.As shown in Figure 3, compared with the C/D ratio of CsA alone, 85.7% of patients had an increased C/D ratio with POS co-medication, the median increase C/D ratio of CsA during co-administration with POS was 0.63 (range, 0.076-1.84(ng/mL)/(mg/kg), P < 0.05).Further, 88.9% of patients had an increased in C/D ratio with FCZ co-medication, the median increase C/D ratio of CsA was 0.42 (range, 0.04-1.06(ng/mL)/(mg/kg), P < 0.001).Similar to the results reported by previous studies [29][30][31].However, the mechanisms underlying the individual variability in the magnitude of the drug interaction between azoles and CsA have yet to be elucidated.In considering the possible mechanisms, we analysed the following: (i) the pharmacokinetic variability of triazole    antifungal agents in the human body is large, and is afected by many factors, such as diet, drug interaction, and the physiological and pathological state of patients and (ii) CYP3A4 gene polymorphism and CYP3A4 enzyme activity.Te wide interindividual variability in drug interactions observed in our study highlights the importance of individualised therapy in clinical practice.Tis interindividual variability strongly suggests that the uniform dose reduction of CsA upon initiating azoles is inappropriate.Terefore, it is suggested that the concentration of CsA should be regularly monitored during the co-administration of the two drugs, and the dose of CsA should be adjusted according to the patient's condition.However, it is yet to be clarifed how long it takes for the interaction between CsA and azoles to reach a steady state after co-administration.Our study further investigated the trend of CsA levels over time after triazole antifungal drugs initiation.We divided the patients into two subgroups based on the co-administration time during 1-7 d and 8-21 d.Our research showed that in both the POS and FCZ groups, the CsA C/D ratio was higher during co-administration (8-21 d) than that during coadministration (1-7 d).Tis may be due to the half-life of these drugs.Previous research has shown that the infuence of POS on CsA levels is associated with the steady state of POS, which is considered to be 8 days according to the halflife of POS.POS reaches the steady-state blood concentration in 7-10 d [32].Sánchez-Ortega et al. [20] results are consistent; the results showed that the allogeneic haematopoietic stem cell transplantation co-administered with POS had an interaction with CsA after 7 days CsA signifcantly increased blood concentrations.Te FCZ prescribing information also indicated that FCZ reached a stable state after 7 days.Hence, we recommend frequent monitoring of CsA concentrations when combined with triazole antifungals.It is particularly crucial to do this after 8 days, preventing excessive concentration from causing adverse reactions.
In keeping with this efect on blood CsA levels and the basis of clinical criteria, the daily dose of CsA was adjusted during co-administration with triazole antifungal drugs.Some studies have recommended an initial empirical reduction in the CsA dose to maintain CsA concentrations within the target range.Te POS prescribing information suggests that CsA should be reduced by 29% at the initiation of combined treatment with POS, but these results were from a study of only four cardiac transplant recipients, and whether this is appropriate for AA remains to be investigated.Robinson et al. [24] conducted a retrospective audit of 29 patients who underwent their frst allograft, and a prospective review of the subsequent cohort of patients who underwent allogeneic HSCT commenced at a lower dose.Tey recommended an initial empirical CsA dose reduction of 30%-40%.Another study on drug interactions with azole antifungal agents concluded that, when starting azole therapy, a pre-emptive dosage reduction of immunosuppressive agents (cyclosporine, tacrolimus, or sirolimus) should be strongly considered [17].Tis fnding is consistent with the results of many studies [33][34][35][36][37].However, some studies do not recommend prereduction of the CsA dose prior to co-administration.Sanchez-Ortega et al. [20] suggested that reducing the CsA dose may lead to a subtherapeutic CsA concentration, thus afecting the therapeutic efect.Hadjibabaie et al. [26] suggested avoiding uniform reduction in all patients before voriconazole administration, and recommend frequent CsA blood concentration monitoring, once voriconazole antifungal therapy is initiated.In addition, a study also indicated that the dose adjustment of CsA inhibitors for initiating azoles should not be decided uniformly but should be determined individually by closely monitoring their blood concentrations [31].Our data show that, when co-administered with POS or FCZ, the reduction of the dose of co-administered CsA (8-21 d) was higher than that of co-administered CsA (1-7 d).Moreover, the dose reduction was greater with POS (8-21 d) than with FCZ (8-21 d).None of the patients in our study received a prereduced CsA dose when co-administered with triazole antifungal agents.Te CsA dose for most patients was adjusted according to the monitored results, which were within the therapeutic window (150-250) ng/mL.It seems cautious not to automatically reduce the dose in advance [38][39][40][41].Terefore, it is preferable to adjust the CsA dose through therapeutic drug monitoring.We recommend that the dose Journal of Clinical Pharmacy and Terapeutics of CsA be further adjusted according to the monitored results when co-administered with triazole antifungal agents.To adjust the dose of CsA in clinical practice, the blood concentration of CsA and the type of co-administered azoles should be considered.
In our study, no serious adverse reactions were found in AA patients.On the one hand, it is possible that CsA concentrations were regularly monitored and the dose was adjusted accordingly; on the other hand, although CsA concentrations increased, they did not exceed the reference range.As well as in a few patients, the concentrations exceeded the reference range, but the dose was not significantly reduced.Tis demonstrated that there is obvious individual variability in the use of CsA in combination with azoles again.Although there were no adverse efects to patients in this study, it is important to be alert to the adverse efects, such as nephrotoxicity, caused by the high concentration of CsA.
Tere are several potential limitations in our study.Statistical analysis could not be carried out because of the small sample size of the combined voriconazole group.In addition, we considered that the magnitude of drug interaction was correlated with plasma concentration of azoles.Due to the retrospective study, concentration of triazole antifungal drugs was not measured.Te infuence of the concentration of azole drugs on the concentration of CsA needs further study.

Conclusions
Tere was signifcant interpatient variability in the magnitude of CsA blood concentration increments after azole coadministration.Terefore, we recommend CsA level monitoring as soon as possible after start of co-administration and dose adjustment on an individual basis.POS had a greater efect on the whole blood concentration of CsA than FCZ in AA patients.When adjusting the dose of CsA in clinical practice, the plasma concentration of CsA and type of coadministered triazole antifungal agents should be considered.

Figure 2 :
Figure 2: Efect of co-administered use of FCZ (a) or POS (b) on the C/D ratio of CsA, and in the FCZ group (c) and in the POS group (d) combined administration time on C/D of CsA in patients with aplastic anaemia.* P < 0.05, * * P < 0.01, * * * P < 0.001.

a
P values for comparisons between with azole co-medication (total) and without azole co-medication.b P values for comparisons between with azole co-medication (1-7 d) and without azole co-medication.c P values for comparisons between with azole co-medication (8-21 d) and without azole co-medication.

Figure 3 :
Figure 3: Individual values of the CsA C/D ratio with or without the co-medication of triazole antifungal agents.* P < 0.05, * * * P < 0.001.

Table 1 :
Patients' demographic data and clinical characteristics.

Table 2 :
Efect of POS or FCZ on C/D ratio of CsA in aplastic anaemia patients.

Table 3 :
Efect of co-administration with triazole antifungal agent drugs on the dose of CsA.

Table 4 :
Laboratory safety data in aplastic anaemia patients without triazole antifungal agent co-medication and after triazole antifungal agent co-medication. is the comparison of biochemical indexes of patients before and after co-administered with triazole antifungal agents. P