Drug-drug interactions of immunosuppressants and other drugs in kidney post-transplant recipients

Introduction: Immunosuppressants (ISS) are the most crucial tools used in the therapeutic regimens of transplant recipients. Nevertheless, these drugs are not the only ones adopted by patients; therefore, knowing the possible drug-drug interactions (DDIs) between immunosuppressants and other drugs commonly used in kidney transplant recipients is essential to ensure the effectiveness and safety of treatments. In this way, the objective is analyzing the DDIs between the immunosuppressants and other commonly used medications on kidney transplant adult recipients with active medical records undergoing post-transplant follow-up for 4.4 years (mean). Methods: First, we performed a cross-sectional study based on patients’ records, in which the patient’s profile and drugs used were examined, and after we analyzed DDIs by the Micromedex Drug Interactions® database. Results: We analyzed 176 patients with a mean age of 47.6(± 12.5); most were male (67.7%), and the majority received a kidney from a deceased donor (81.4%). Patients were exposed to 15.0 (± 5.4) different medicines after the transplantation, and 7.4 (± 4.0) of these medicines were simultaneous. After analyzing the DDIs according to the severity of interaction, documentation quality interaction effect, clinical management and probable interaction mechanism, the most frequent interaction was with tacrolimus, classified as moderate, and the 3 major causes of interaction occurred with azathioprine according to the Micromedex database. The primary medicines involved with immunosuppressant interactions were proton pump inhibitors, ranitidine, domperidone, amlodipine, enalapril, allopurinol, cyclobenzaprine, amitriptyline, fluoxetine, and ciprofloxacin. These DDIs’ effects were related to, mainly, increase their immunosuppressant activity. Conclusion: Although the immunosuppressants analyzed lacked many clinical DDIs significance with other medicines, the healthcare team needs to monitor their DDIs’ effects to prevent and minimize side effects in transplanted recipients.


INTRODUCTION
Organ transplantation has been rising since the renowned Herrick brothers' transplant in 1954 1 .Since the first medicine many new treatments and improvements have led to a better recovery, organ acceptance, fewer rejections, and a better quality of life 1 .
Brazil has its own national organ transplantation program, part of the Brazilian Public Unified Health System that offers free universal coverage, responsible for over 95% of the transplantations made in the country.Brazil has the most extensive worldwide public system in kidney transplantation, with 58,293 kidney transplants performed between 2011 and 2021 2,3 .
Immunosuppressants are the cornerstone drugs for transplant recipients, used not just to prevent acute organ rejection, but also to treat other diseases such as cancer and autoimmune diseases.Understanding the T cell's three signal model is essential to comprehending immunosuppressor's action mechanisms 4,5 .Alloimmune response initiates with an antigen-presenting cell (APC) presenting a donor antigen triggering T cell receptors (TCR/CD3) of naïve and memory T cells (signal 1) and a co-stimulated APC's CD80 (B7-1), CD86 (B7-2) or CD80/86 (B7) complexes engaging CD28 on T cells (signal 2).These first two signals' completion activates three transduction pathways (the calcium-calcineurin pathway, the RAS-mitogen-activated protein MAP kinase pathway, and the nuclear factor-κΒ pathway), resulting in the expression of different molecules, such as interleukin-2 (IL-2), CD154, and CD25.IL-2 and IL-15 stimulate T cells receptors (signal 3), unchaining the mammalian target of rapamycin (mTOR) pathway, which triggers cell proliferation, and after nucleotide synthesis, T cells shift into effector phenotype 4,6,7 .
Among drugs used in graft maintenance, there are calcineurin inhibitors, mTOR inhibitors, antimetabolic agents, nucleotide synthesis inhibitors, and glucocorticoids.Calcineurin inhibitors (tacrolimus and cyclosporine) inhibit calcineurin, not allowing IL-2 transcription, suppressing T cell and T cell-dependent B cell activation.Mycophenolate sodium (enteric-coated mycophenolate) inhibits guanosine monophosphate nucleotides and blocks purine synthesis, preventing the proliferation of T and B cells.Azathioprine is a nucleotide synthesis (DNA, RNA) and protein synthesis inhibitor.Glucocorticoids hinder several cytokine encoding genes, particularly IL-2 4,5 .
Diabetes, hypertension, and dyslipidemia often co-occurs in chronic kidney disease patients and persist after kidney transplantation.With the multidrug immunosuppression strategy, knowing possible drug-drug interactions (DDIs) between the immunosuppressants and drugs used is crucial to treating comorbidities in the post-transplant follow-up period.The most common chronic diseases that lead to kidney transplantation can also be developed through the continuous use of immunosuppressants, are hypertension, diabetes mellitus, and dyslipidemia.Managing these chronic diseases and the immunosuppression in the clinical environment may become challenging due to the number of medicines used simultaneously.Nonetheless, DDIs analysis is not always a priority of the healthcare team.The long-term effects of immunosuppressants and their interaction with other commonly used drugs must likewise be considered in calculating the ideal immunosuppressant dose to prevent graft rejection while avoiding drug-specific complications and the risk of developing opportunistic infections or neoplasm occurrence [7][8][9][10] .
This article reviews the DDIs between the most taken immunosuppressants and other commonly used medicines in the kidney transplant recipients from patients' records at the Brazilian Transplant Center, Brasília-DF, Brazil.

METHODS
This observational and cross-sectional study recruited patients from the outpatient clinic of a Brazilian Transplant Center located in Brasília, the capital of Brazil, connected to Brasília's University Hospital (HUB) and licensed for kidney and corneal transplants.We collected the epidemiological data in 2019 for all patients with active medical records in December of 2018, excluding those who died, were transferred to other care teams, were transplanted by other teams, pediatric patients and patients without physical records.
We performed data extraction from patients' records and analyzed drug-drug interactions (DDIs) from the IBM Micromedex Drug Interaction ® database 11 and this project was approved by the Ethical Committee (CAEE 02637918.0.0000.8093).
Demographic data evaluated were age, gender, underlying renal disease, comorbidities, the time of follow-up, and type of donor.Data on the medicines used in ambulatorial care was also gathered.We investigated all medicines prescribed in the patients' records and used the Anatomical Therapeutic Chemical classification with Defined Daily Doses (ATC/DDD) index for the anatomic, chemical, and therapeutic drug classification 12 .
We also analyzed each patient's medication list in the IBM Micromedex Drug Int ® database for harmful interactions.The analyzed DDIs data were collected and organized in the severity of the interaction, documentation quality, interaction effect, clinical management (if the drug needs to be used simultaneously), and probable interaction mechanism.
The IBM Micromedex Drug Int ® database classifies the DDI severity into five categories: contraindicated, major, moderate, mild, and unknown.Contraindicated drugs cannot be used concomitantly.Major DDI classification could represent life risk or may require medical intervention to diminish or avoid serious adverse effects, or both.Moderate interaction includes a possible exacerbation of the patient's base disease, a requirement of alteration (dosage)/change in their current medication, or both.Mild DDI classification may result in limited clinical effects that generally do not require changing the actual treatment.
The documentation quality was classified as excellent, good, poor, and unknown.Excellent documentation has controlled studies that clearly established the interaction's existence.Good documentation suggests that the interaction exists but lacks controlled studies to support this interaction.Poor documentation explains that the information available is unsatisfactory, though pharmacologic considerations lead the clinicians to suspect a possible interaction or that the information given is suitable for a similar drug.Finally, unknown means a lack of both the severity and documentation on the nature of the interaction and documents supporting the said interaction 11 .We organized the data in an Excel database and used descriptive analysis to present the results.The quantitative variables were presented as mean, median, and standard deviation and the qualitative variables were shown in percentage.

RESULTS
We analyzed 167 from 253 patients who received a renal transplant in the Brazilian Transplant Center at the HUB (Brasília University Hospital) and had active medical records in December of 2018, exclusion criteria can be consulted on methods.
In this health care unit, most patients were men (67.7%) and received the organ from deceased donors (81.4%).The total analyzed patients had a mean age of 47.6 (± 12.5) and a median age of 48 years.
The most common causes of chronic kidney disease (CKD) were chronic diseases such as hypertension (13.2%) and diabetes (10.2%), though often, the CKD cause is undetermined (34.7%).The patients included in the study were undergoing post-transplant follow-up for 4.4 (± 2.7) years (Table 1).The most common health problems identified in our research before and after transplantation were hypertension and diabetes mellitus (Table 1).
The most common classes of pharmaceuticals are presented in Figure 1 and detailed in Table 2. Clinicians prescribed seven different immunosuppressants and 265 other drugs in this healthcare unit, and patients were exposed to 15.0 (± 5.4) different medicine at home during this period after the transplantation and had a mean of 7.4 (± 4.0) simultaneously taken medicines.
The most used calcineurin inhibitor was tacrolimus (97%).Despite the lower use of cyclosporine in this center, some medical interactions involving this immunosuppressant were also found (Figure 2).Among these drugs, thirty-four ferent possible drug-drug interactions (DDIs) between immunosuppressants and other drugs were identified, and only one was classified as contraindicated (Table 3).Seventy-five DDIs between non-immunosuppressive drugs were also found but were not included in this study.Among the immunosuppressants, most possible DDIs were related to tacrolimus and azathioprine.These DDIs commonly decrease the drug's effectiveness or safety by reducing their action or potentiating adverse effects, respectively.The other pharmacological classes involved with DDIs were quite diverse, including antimicrobials, anti-ulcers, antihypertensives, and central action drugs.Most described DDIs management was dose reduction or immunosuppressant dose monitoring.*At least one prescription.A: Alimentary tract and metabolism; B: Blood and blood-forming organs; C: Cardiovascular system; D: Dermatological; G: Genito urinary system and sex hormones; H: Systemic hormonal preparations, excl.sex hormones and insulins; J: Anti-infective for systemic use; L: Antineoplastic and immunomodulating agents; M: Musculo-skeletal system; N: Nervous system; P -Antiparasitic products, insecticides, and repellents; R: Respiratory system; S: Sensory organs; V: Various; X: Unclassified, such as phytotherapies.Source: Brito 13 .
Table 2: Frequency of the main non-immunosuppressive drugs used, by the anatomical, therapeutic chemical (ATC) classification, in renal transplant patients in the university hospital in Brasília (HUB), 2018.

Blood and Blood-forming organs
Salicylic acid ▲ ▲
The three major DDIs occurred with azathioprine.The first DDI with allopurinol causes toxicity and requires reducing the dose to ¼ of azathioprine.The second DDI with enalapril maleate induces myelosuppression and requires close monitoring if coadministration is needed.Finally, the last DDI with sulfamethoxazole/trimethoprim augments the risk of bone marrow suppression, especially in renal transplants (Supplement 1).
Tacrolimus had twelve DDIs with other drugs found (Table 3), eleven major and one moderate.It interacted with proton-pump inhibitors, such as amlodipine, ranitidine hydrochloride, and ketoconazole, resulting in increased tacrolimus exposure (Supplement 1).
Both cyclobenzaprine and domperidone showed a high risk of QT-interval prolongation when combined with tacrolimus.Prednisone may decrease tacrolimus levels if used together and, thus, increase the risk of organ rejection.As for the documentation quality, it was considered regular in most situations (61.5%).

DISCUSSION
Tacrolimus is the most prescribed immunosuppressive drug in combination with sirolimus in the transplant service where this study was conducted.In Vitko et al.'s 14 multicenter study, the tacrolimussirolimus combination regimen showed a significantly lower incidence of biopsy-proven acute rejection compared with other therapeutic regimens, such as tacrolimus-mycophenolate or cyclosporin-sirolimus.Nonetheless, the tacrolimus-sirolimus regimen also showed elevated total serum cholesterol levels; this elevation can be attributable solely to sirolimus, and if statins treatment does not normalize serum cholesterol levels, reducing sirolimus dose without compromising the immunosuppressive efficacy might be a possible solution 14 .
Patients who undergo kidney transplantation surgery have at least a previously developed chronic kidney disease (CKD), generally accompanied by other comorbidities such as hypertension or diabetes, or sometimes both.These underlining conditions result in a considerable quantity of drugs per patient, which, in most cases, is the only way to maintain their comorbidities stabilized.Noticeably, when analyzing immunosuppressants' interactions with the most common drugs used in kidney transplanted patients, the immunosuppressants from the antimetabolite agents and the calcineurin inhibitors families showed the most drug-drug interactions (DDIs) [15][16][17] .
The immunosuppressants are used in higher doses at the beginning of the post-transplant until the highest risk of graft rejection surpasses.Once this period passes, the dosage of these drugs will drop until reaching a maintenance level that would probably last their entire life.In most cases, monitoring of pharmacological interactions is essential as DDIs are not always dose-dependent and have the possibility of occurring when a lower dose is administered 18 .
Our patients' profiles included gender, age, base disease, the time within the renal service, and type of donor.Similar to Ribeiro et al. 19 study in which 66.7% of the recipients were male, the majority of patients were male (67%).This prevalence toward male recipients is expected as one of the most important causes of renal transplant is CKD, which is more common in males than females.Luvisotto, Carvalho, and Galdeano 20 considered that all transplant recipients' base disease was CKD and that 49% of the patients were diagnosed with hypertension and 20% with diabetes was due to this base disease.
Polypharmacy in transplanted kidney recipients is not just correlated to immunosuppressive therapy but also with other drugs used for health problems such as diabetes mellitus, hypertension, and primary kidney diseases and for the prophylaxis protocols such as the use of sulfamethoxazole-trimethoprim and omeprazole.Importantly, drugs such as nystatin, albendazole, and ivermectin are in the list of medicines used for prophylaxis in the immediate post-transplant process, which is why they were not included in the most used drugs' list in the ambulatory service 21 .
One of the most adverse DDIs with azathioprine is with xanthine oxidase activity inhibitors (e.g., allopurinol), as xanthine oxidase takes part in azathioprine metabolism.This combination might cause severe leukopenia; hence it should be avoided.If the combination must be used, careful monitoring of the white blood cell count becomes necessary 22 .
Studies revealed that some drugs could increase blood levels when combined with immunosuppressants, such as calcineurin inhibitors, tacrolimus, and cyclosporine.We found in the Micromedex database that the recommendation for calcium channel blockers was to monitor doses carefully for higher concentrations or toxicity of the immunosuppressant.A singledose study from China evaluated the CYP3A45*3 allele's effects on the tacrolimus and amlodipine pharmacokinetics and established significant DDIs involving the CYP3A45*3 between these two drugs 23 .
According to our database research, the interaction between allopurinol and azathioprine could be monitored by reducing the immunosuppressant dose.Even though research evidence recommends that this combination be avoided as the outcomes from this interaction are not secure 24 .
Another important interaction unidentified by our database is between ganciclovir and mycophenolate, used for treating Cytomegalovirus.Although the concentration in which these two drugs are used together has not shown significance, it is essential to evaluate other potential adverse effects, especially their gastrointestinal effects 25 .
Another unidentified drug-drug interaction in our Micromedex database study was the interaction between isoniazid and immunosuppressants used for tuberculosis treatment, one of the most important opportunistic infections in transplant recipients.Explained in several studies as possible hepatotoxicity, this case type of interaction is moderate and causes elevated levels of immunosuppressants, needing laboratory monitoring for dose adjustment.The absence of this interaction in our sample study could be explained by a low prevalence of tuberculosis in the local healthcare service and the triage treatment protocol for all patients on the transplants list 26 .
In renal transplanted patients, the number of medications and their dose is high in the first months after surgery to prevent graft rejection.Immunosuppressants are the main drugs part of the transplanted patients' primary treatment schedule that interact with many drugs also used in these patients' treatment, such as antihypertensives, anti-acids, anti-inflammatory, and other drugs.
Immunosuppressive therapy maintenance is the most critical step in the transplant puzzle for successful organ transplantation.Consequently, knowing the possible interactions of immunosuppressive drugs with other drugs used by the recipient aids in providing the most suitable drug combination to keep the patients' new organ functionating while decreasing the drugs' side-effects, thus providing them a better life quality.The immunosuppression in organ transplantation prevents graft rejection and helps minimize undesired side effects such as infection, malignancy, and drug toxicity.It is not uncommon for kidney recipients to take two immunosuppressants concurrently in their daily routine for extended periods; some must keep taking at least two immunosuppressants for their entire lifetime.When prescribing two immunosuppressants, knowing the best combinations and avoiding the least compatible ones is vital for clinicians.For this, it is essential to combine two different classes of immunosuppressants and a glucocorticoid, most frequently prednisone.DDIs' clinical impact involves both pharmacokinetics and pharmacodynamic effects reflected in the clinical observation and changes in the medications' serum concentrations.In this context, DDIs can favor clinical treatment, and by knowing and learning their effects, the healthcare team can properly establish appropriate drug management 27 .
Our study has some limitations.As an observational study, it might be missing clinical experience data from the transplant service and their most common DDIs involving immunosuppressants, as well as missing DDIs that went undetected in the IBM Micromedex ® Drug Int database.Furthermore, it is a single institution study with a small sample size that involved only drugs that outpatients use.
It is worth mentioning that this is preliminary work and must be updated in short periods to ensure that it is updated with the current drug combinations protocol used in the different care units.
It is fundamental for all health providers to understand the drug-drug interaction (DDIs) of the drugs they prescribe their patients.This understanding and access to different tools (e.g., IBM Micromedex ® Drug Int) enable health providers to determine the most suitable for their patient's specific profile from the possible drug combinations available, thus helping prevent or minimize adverse effects.
Our work analyzed the Brazilian Transplant Center at the HUB (Brasilia University Hospital) ambulatory drug use list determining the most critical medical interactions between immunosuppressants and other drugs used by the renal transplanted outpatients.SUPPLEMENT 1   Immunosuppressants and their drug interactions with the most common drugs used in the transplant center at the HUB (Brasilia University Hospital).

Figure 1 :
Figure 1: Drugs used* by transplant patients according to the leading anatomical group of the Anatomical, Therapeutic, and Chemical Classification (ATC), 2018 (167 patients).

Table 3 :
Immunosuppressants and their drug-drug interactions (DDIs) with the commonly used non-immunosuppressive drugs in post-kidney transplant patients based on IBM Micromedex ® database.