Multicentre, controlled, randomised and single-blind, adaptive phase IV protocol to evaluate effectiveness and cost-effectiveness of a pre-emptive genotyping strategy to optimise tacrolimus dosage in a pretransplant chronic kidney disease population cohort: iPHARMGx TRANSPGx nested clinical trial

Abstract

Introduction

Genetic variations impact drug response, driving the need for personalised medicine through pre-emptive pharmacogenetic testing. However, the adoption of pre-emptive pharmacogenetic testing for commonly prescribed drugs, such as tacrolimus, outside of tertiary hospitals is limited due to a lack of pharmacoeconomic evidence to support widespread implementation by healthcare policymakers. The iPHARMGx Consortium addresses this by developing the TRANSPGx clinical trial to assess the hypothesis that widespread adoption of a pre-emptive genotyping scheme in populations susceptible to receiving tacrolimus as immunosuppressive therapy following a kidney transplant is effective, cost-effective and feasible within the Spanish National Health System (SNHS) when compared to the standard of care tacrolimus dosing.

Methods and analysis

The TRANSPGx trial is a multicentre, adaptive, randomised, controlled, pragmatic phase IV clinical trial nested within the iPHARMGx master protocol, with two parallel arms, aiming for superiority. Randomisation will be conducted on an individual basis with a centralised approach, with stratification by centre. After inclusion in the trial and completion of genotyping, subjects will be randomly allocated to either the experimental group (pharmacogenetic genotype-guided tacrolimus prescription) or the standard of care tacrolimus prescription (as deemed by the attending physician). The primary objective is to assess the effectiveness of a tacrolimus pre-emptive genotyping strategy in reaching tacrolimus target plasma concentrations after renal transplant. A total of 114 subjects will be recruited among the different participating centres, provided that no futility/efficacy boundary is reached in the prespecified interim analyses. Recruitment will be carried out during a 12-month period, and subjects will be followed for a 6-month period.

Ethics and dissemination

The TRANSPGx trial received ethical approval on 16 January 2025 (La Paz University Hospital 2024.740). Results will be disseminated via publication in peer-reviewed journals as well as presentation at international conferences. Trial results will be submitted for publication in an open access peer-reviewed medical speciality-specific publication. Trial registration of this study can be located at both the EU Clinical Trials Register, available from https://euclinicaltrials.eu/search-for-clinical-trials/?lang=en and https://clinicaltrials.gov. Registration on both websites was done before the enrolment of the first patient complying with European regulations. The EU Clinical Trials Register is a primary registry according to the WHO.

Trial registration number

EU CT number: 2024–5 16 596-32−00/Clinical trial Identifier (ClinicalTrials.gov): NCT06701825. Protocol V. 1.2, 8 January 2025.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• The study will be randomised and stratified by centre, minimising selection bias.

• The adaptive design allows for interim analyses and the possibility of early stopping for efficacy or futility, improving trial efficiency without requiring the full sample size.

• The single-blind design may introduce some risk of bias, although this is partially mitigated by the use of objective endpoints and standardised procedures.

• The pragmatic nature of the trial, while enhancing external validity, may introduce variability in clinical practice across centres, which could influence secondary outcomes. This design may reduce the difficulties in recruiting subjects.

• The relatively short follow-up period (26 weeks) limits the ability to assess long-term clinical and economic outcomes, such as chronic graft function or patient survival.

Introduction to the TRANSPGx trial

Chronic kidney disease is an increasingly prevalent condition, affecting up to 16% of the adult population in several continents.¹ Renal transplant remains the treatment of choice for patients with end-stage renal disease, providing substantially longer survival and better quality of life than dialysis. In addition, pre-emptive transplantation is associated with better outcomes than transplantation performed after dialysis initiation.² Immunosuppression following kidney transplantation aims to prevent and treat acute rejection and minimise the risks of graft loss and death.³

Tacrolimus stands as the mainstay of current immunosuppressive drug therapy after solid organ transplantation. However, its clinical implementation has been challenging due to its narrow therapeutic range and highly variable pharmacokinetics.⁴ Despite the adjustment of the initial dosage based on body weight and the ongoing monitoring of drug blood concentrations, achieving the desired target concentrations proves to be a slow process marked by a significant degree of inter-individual and intra-individual variability.⁵ The oral bioavailability is limited (average of 20–25 per cent) as a result of poor absorption, partial metabolism by enzymes in the bowel mucosa, intestinal P-glycoprotein (P-gp) efflux and first-pass hepatic metabolism.⁶ For this reason, the trough level of tacrolimus is currently used for routine monitoring after kidney transplantation.⁷

Tacrolimus is metabolised via cytochrome P450 3A (CYP3A) enzymes (mainly CYP3A4 and CYP3A5) in the liver and upper gastrointestinal tract.³ Variability has been attributed to genetic polymorphism of these metabolising enzymes, with single-nucleotide variants (SNVs) being the main type of variations involved.⁸ Compared with CYP3A5, the catalytic efficiency of CYP3A4 was relatively low.⁸

This evidence suggests that the expression and/or function of CYP3A5, CYP3A4 and P-gp are closely related to the PK of tacrolimus. Therefore, genetic polymorphism of these proteins, or factors that have an impact on them, can influence bioavailability and lead to inter-individual differences.⁹

As stated before, CYP3A5 may play a more dominant role in the metabolism of tacrolimus than CYP3A4.⁸ A single nucleotide polymorphism (SNP) in the CYP3A5 gene involving an A to G transition within intron 3 (rs776746) was strongly associated with CYP3A5 protein expression. Individuals carrying at least one CYP3A5*1 allele (considered to be the wild-type allele, corresponding with an A at position 6986 of CYP3A5) were found to express large amounts of CYP3A5 protein, whereas individuals homozygous for the CYP3A5*3 allele did not express significant quantities of CYP3A5 protein. The CYP3A5*3 allele causes alternative splicing, resulting in protein truncation and a severe decrease of functional CYP3A5.^{10 11} This positive correlation between CYP3A5 genotypes and tacrolimus pharmacokinetics has been stated as well in the paediatric population.¹²

CYP3A5 has shown consistent evidence on its significant influence on drug metabolism. Individuals carrying at least one functional CYP3A5*1 allele exhibit increased tacrolimus clearance and require higher doses to achieve therapeutic trough concentrations compared with non-expressors (*3/3 genotype). This association has been confirmed in multiple studies, including a meta-analysis by Rojas et al⁵ and establishes the basis for the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines,¹³ which currently provide dosing recommendations for CYP3A5. In contrast, other genetic variants such as CYP3A422 and ABCB1 polymorphisms have shown more modest or inconsistent effects on tacrolimus exposure, and to date, no formal dosing guidelines have been established for these loci.

Early achievement of tacrolimus therapeutic levels is recognised as an important determinant of transplant outcomes. Recent evidence indicates that kidney transplant recipients who reach tacrolimus trough concentrations above 7 ng/mL within the first week post-transplant experience significantly lower rates of acute rejection in the first month.¹⁴ In contrast, prolonged subtherapeutic tacrolimus exposure has been associated with higher rejection risk and increased pharmacokinetic variability, which itself is a known risk factor for rejection.⁷ Rapid attainment of target tacrolimus levels may also reduce periods of acute overexposure, potentially lowering the risk of calcineurin inhibitor-related nephrotoxicity. Therefore, the day 4 trough concentration (target 7–10 ng/mL) is a meaningful surrogate endpoint, reflecting early immunosuppressive control that may be linked to clinically relevant outcomes

Pre-emptive pharmacogenetic testing is gaining prominence in the rapidly advancing field of personalised medicine as a means of individualising treatment plans, enhancing treatment effectiveness, and mitigating the occurrence of adverse events. For this reason, over the past few decades, regulatory agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), in collaboration with scientific societies, have steadily incorporated genetic profiles associated with higher incidences of ADRs to the labels and guidelines of widely prescribed medications. These updates, supported by evidence related to relevant enzyme-encoding genes, provide new information regarding the impact of genetic variations on determining safer and more optimal dosages.

Despite the potential benefits of pre-emptive pharmacogenomics, several limitations still hinder its implementation beyond a limited number of tertiary hospitals within healthcare systems.¹⁵

One major challenge is the lack of pharmacoeconomic studies evaluating the balance between the costs and effects of pre-emptive pharmacogenomics.¹⁵ While pre-emptive pharmacogenetic testing shows promise in improving patient outcomes and reducing the occurrence of adverse drug reactions, the lack of clear evidence on its cost-effectiveness remains a significant challenge for its widespread adoption.¹⁶ Despite the development of comprehensive clinical guidelines and the inclusion of pharmacogenetic information in drug labels, the current state of pharmacogenomic implementation in healthcare systems and communities shows that most testing remains confined to tertiary or highly specialised hospitals, likely due to patient characteristics.¹⁷ This highlights the need for further research that better establishes the efficacy, cost-effectiveness and feasibility of pre-emptive pharmacogenomic testing for more commonly prescribed drugs in both inpatient and outpatient settings. Such research will enable healthcare policymakers and regulators to make informed decisions about the broader integration of pharmacogenomics into standard clinical practice and to fully endorse its use.^{16 18}

To address these challenges, the IPHARMGx Consortium has been formed, comprising 11 hospitals across the Spanish National Health System (Gregorio Marañón General University Hospital, Central Defense Hospital Gomez Ulla, General Hospital of Tomelloso, General University Hospital of Alicante, University Hospital of Burgos, University Hospital of the Canary Islands, University Hospital Germans Trias i Pujol, University Hospital Marqués de Valdecilla, University Hospital Virgen de la Victoria, University Hospital La Princesa and University Hospital La Paz), the Autónoma de Madrid University and the Health Technologies Assessment Agency of Instituto de Salud Carlos III. This consortium has received funding from the ISCIII within the framework of a public call for personalised medicine (PMP22/00055).

Methods and analysis

This study protocol, in conjunction with the iPHARMGx master protocol,¹⁹ follows the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines (online supplemental material 1).²⁰

Hypothesis and design

The overall goal of all trials nested within the iPHARMGx framework is to evaluate pre-emptive genotyping strategies in high-risk populations susceptible of receiving certain targeted treatments in terms of both efficacy and cost-effectiveness. Our hypothesis is that pre-emptive genotyping in populations at risk is efficacious, cost-effective, and feasible within the Spanish National Health System when compared with the current standard of care. Nested studies will be pragmatic, adaptive, phase IV, multicentre, randomised, parallel clinical trials. The following eligibility criteria will be applicable to all trials nested within the iPHARMGx Platform:

• All nested trials must have at least one clearly defined pharmacogenetic biomarker present in high-risk populations with an IA level of evidence.

• All nested trial populations must have a target genetic biomarker that has been demonstrated as actionable according to clinical practice guidelines for the treatment of the medical condition being investigated in the study.

All nested trials must have a clearly defined efficacy outcome or a validated efficacy surrogate outcome.

TRANSPGx study objectives

Primary objectives

• To assess the effectiveness of a pre-emptive pharmacogenetic strategy in reaching tacrolimus target plasma concentrations.

Secondary objectives

• A pharmacoeconomic analysis to assess the feasibility of implementing a preemptive pharmacogenetic strategy in reducing renal transplant rejection and the incidence and severity of adverse events (AEs).

• To evaluate the safety and tolerability of tacrolimus treatment guided by a preemptive pharmacogenetic strategy.

Exploratory objectives

• Identification of new actionable genes/relevant polymorphisms within the predefined population subsets.

• The assessment of novel prognostic and predictive genetic biomarkers related to tacrolimus safety and effectiveness. This assessment will be performed using techniques available at the National Centre for Oncological Investigations (CNIO) and through genome-wide association studies, when applicable.

Inclusion criteria

Each potential participant must satisfy all of the following criteria to be enrolled in the study:

1. Participants must be willing and able to provide written informed consent prior to the initiation of any study procedures.

2. Subject or their legally authorised representative has voluntarily signed the informed consent document.

3. Participant is on the waiting list for a kidney transplant.

4. Subject is able and willing to participate and be followed for the majority of the study duration and is able to adhere to the procedures specified in the protocol.

Subjects must be naïve to any genotyping test of the following genes: CYP3A5.

Exclusion criteria

Any potential participant who meets any of the following criteria will be excluded from participating in the study:

1. Known hypersensitivity/allergy reaction to tacrolimus or any of the excipients.

2. History of renal, heart and/or liver transplant.

3. History or clinical evidence of any disease and/or existence of any surgical or medical condition, which might interfere in a relevant manner with the absorption, distribution, metabolism or excretion of the study treatment, except for renal disease.

4. Any condition or situation precluding or interfering with compliance with the protocol.

Any condition, at the investigator’s discretion, for which renal transplantation and/or study treatment should not be received.

Study procedures

All study procedures and the schedule of assessments are summarised in table 1.

Table 1. Study procedures and the schedule of assessments.

Visit	Visit 0: screening	Visit 1 baseline	Visit 2: effectiveness evaluation	Visit 3	Visit 4	Visit 5: end of study
Day	Days −180 to −14	Day 0	Day 4*	Week 4†	Week 15†	Week 26†
Eligibility check	X
Informed consent	X
Medical history/baseline medication	X
Concomitant medication		X	X	X	X	X
Randomisation		X
Physical examination	X‡		X§	X§	X§	X§
Vital signs¶	X		X	X	X	X
Pharmacogenetics blood draw	X
Tacrolimus blood concentration levels			X	X	X	X
Lab determinations**		X	X	X	X	X
Treatment initiation		X
Adverse events evaluation			X	X	X	X

^*+/- 1 (1) +/- 1 day window to be performed.

^†+/-5(2) +/-5 days window to be performed.

^‡At screening visit, physical examination must include height, weight and body mass index (BMI) calculation.

^§At clinician’s discretion.

^¶Vital signs: Heart rate (bpm), blood pressure (mmHg), temperature (°C) and oxygen saturation (%).

^**Laboratory assessments will include complete blood count (CBC), coagulation, serum biochemistry with electrolytes (Na+,K+,Cl-), glucose, complete renal and liver function tests. Urine pregnancy tests may be performed at the investigator’s discretion. Screening laboratory determination will not be required if subject has a lab determination including all required parameters performed for any reason up to 1 month prior to randomisation visit. Additional laboratory determinations can be done following local guidelines.

The following list of procedures will be performed at all visits after inclusion:

1. Eligibility check:

This assessment will involve a comprehensive review of specific inclusion/exclusion criteria outlined in the protocol to ensure that participants can continue to participate.

• 2 1Medical history and physical examination

Participants’ medical histories will be documented, including any relevant pre-existing conditions and medications. Any new information must be recorded in the medical history.

A comprehensive physical examination will be conducted to assess participants’ overall health and monitor changes throughout the study. This examination will include an evaluation of vital signs, such as blood pressure, heart rate, respiratory rate and body temperature, as well as a comprehensive systems and organ examination.

• 3 1Concomitant medication

Any new medication or change in previously recorded medication must be recorded.

Visit 0: screening visit

The study will be explained to the participant, and informed consent (online supplemental material 2) will be obtained prior to any study procedure.

After checking the selection criteria, informing the patient, and obtaining the signed Informed Consent Form (ICF) by the investigator, a sample for pharmacogenetic analysis will be collected. The pharmacogenetic sample should preferably be collected during the screening visit, but it can be collected at any point prior to the initiation of tacrolimus therapy. An aliquot of the DNA sample will be stored at −20°C for quality control purposes and/or further genetic analysis by techniques available at a central laboratory affiliated with the Precision Medicine Infrastructure associated with Science and Technology (IMPaCT).

The subject must have a mandatory screening laboratory analysis, including all safety parameters described in this study, that can be performed between a window of 30 to 1 day before the baseline/randomisation visit. Screening laboratory analyses may be conducted for any reason, as long as they fall within the aforementioned window and can in theory be dated before the signing of the ICF. Therefore, screening laboratory determination will not be required if the subject has had a laboratory determination including all required parameters performed for any reason within 1 month prior to the randomisation visit.

Visit 1: baseline – up to 180 days after first screening visit

If the subject has successfully completed the screening visit and is eligible based on compliance with all inclusion criteria and none of the exclusion criteria, randomisation and baseline procedures may be delayed for up to 6 months after screening. This means that the prescription of tacrolimus can be withheld for a maximum of 6 months in subjects who may not require immunosuppressive therapy but are likely to require it in the foreseeable future.

The subject will be randomised to receive either standard of care treatment (control arm) or pharmacogenetic guideline-based treatment (experimental arm). Before treatment initiation, for subjects allocated to the experimental arm, a dose or treatment recommendation will be provided to the prescribing clinician by a clinical pharmacologist or a pharmacogenetics-expert pharmacist. This recommendation will take into account the subject’s pharmacogenetic profile, clinical background, and any potential drug-drug interactions or phenoconversion risks based on their concomitant medications and medical history. On completion of baseline visit subject will initiate treatment with the prescribed dose.

Visit 2: 4 days after treatment initiation (+/- 1 day window)

Blood samples for laboratory assessments, including assessment of the primary endpoint, will be obtained. Vital signs (including blood pressure, heart rate, respiratory rate and body temperature) must be recorded. All adverse events, whether related or unrelated, must also be documented.

Visit 3: week 4 (+/- 5 days window)

Blood samples for laboratory assessments will be obtained. Vital signs (including blood pressure, heart rate, respiratory rate and body temperature) must be recorded. Related and unrelated adverse events must be recorded.

Visit 4: week 15 (+/- 5 days window)

Blood samples for laboratory assessments will be obtained. Vital signs (including blood pressure, heart rate, respiratory rate, and body temperature) must be recorded. Related and unrelated adverse events must be recorded.

Visit 5: end of study – 26 weeks after treatment initiation (+/- 5 days window)

The end-of-study visit for patients who remain in the study until the last follow-up visit without withdrawal due to therapeutic failure or toxicity will be visit 6 (week 26). For patients who discontinue follow-up before week 26 for any reason, the last face-to-face visit will serve as the end-of-study visit.

Blood samples for laboratory assessments will be obtained. Vital signs (including blood pressure, heart rate, respiratory rate, and body temperature) must be recorded. Related and unrelated adverse events must be recorded and appropriate questionnaires be completed.

Sample size calculation, efficacy and futility boundaries

This is an adaptive trial nested within the iPHARMGx protocol. For the purpose of this clinical study protocol, we have established three distinct stages (denoted as J=3), each representing a critical point at which data analysis will be conducted. Our objective is to maintain a desired level of statistical rigour, with a type I error rate (alpha) set at 0.05 and a type II error rate fixed at 0.2. The third and final stage corresponds to the ultimate analysis.

To power this trial for a meaningful effect size (δ=0.554), we have calculated an estimated total sample size of 102 subjects, with 17 subjects allocated to each arm within each stage. This sample size determination is critical for ensuring the statistical robustness of the trial and the ability to detect the intended therapeutic effect. This total number of subjects has been increased to 114 to factor in possible pre-randomisation loss of subjects (10%) that despite being genotyped do not receive tacrolimus for whatever reason. Therefore, in order to adequately assess the pharmacoeconomic feasibility of the intervention, they must be included in the pharmacoeconomic endpoint analysis.²¹

The rationale behind the sample size calculation per arm per stage is grounded in existing published data. Research indicates that about 44.4% individuals who receive tacrolimus standard dosing reach therapeutic range within 4 days while about 71.4% of individuals with tacrolimus genotype-guided dosing reach therapeutic range.²² These insights from the literature help us in estimating the expected effect size and risk within our target population, allowing us to tailor our trial accordingly. Based on these data and taking the percentages 44.4% and 71.4%, respectively, the effect size is calculated in R statistical software as delta=0.554.

We have established three distinct stages (J=3), each representing a critical point at which data analysis will be conducted. Our objective is to maintain a desired level of statistical rigour, with a type I error rate set at 0.05 and a type II error rate fixed at 0.2. The third and final stage corresponds to the final analysis. Interim analyses will employ O’Brien-Fleming type stopping boundaries to control type I error during the study. These are as follows: (4.443, 2.233, 1.494) for efficacy and (0.358, 0.999, 1.494) for futility. If either assessment exceeds the prespecified threshold, study termination is warranted. These boundaries were calculated using the graphical user interface (GUI) built on (and in to) V. 2.2.0 of the R package OptGS²¹ (figure 1).

Figure 1. Futility and efficacy thresholds plot that must be met in order to warrant study termination.

Randomisation and blinding

This is a randomised single-blind trial; therefore, blinding will be required for only participating subjects. A double-blind design was not considered feasible due to the methodological difficulty of prescribing a dose of tacrolimus that would differ from routine clinical practice conditions. It has been deemed sufficiently appropriate to blind only subjects since the primary endpoint is an objective laboratory assessment (tacrolimus concentration levels).

Subjects will be allocated to either the intervention arm or the control arm at a 1:1 ratio. An intermedia Interactive Web Response Systems (IWRS) software will be used for subject randomisation. Under normal circumstances, the blind should not be broken until all participants have completed the study and the database is finalised/locked. The investigator may, in an emergency, reveal the identity of the intervention to the subject by contacting the IWRS. While the responsibility to break the intervention code to a third party in emergency situations resides solely with the investigator. Telephone contact with the sponsor or its designee will be available 24 hours per day, 7 days per week. Only the data manager and the statistician will have access to the unblinded randomisation list, under the foreseeable circumstances.

In the event the blind is broken, the sponsor must be informed as soon as possible. The date, time and reason for the unblinding must be documented in the appropriate section of the electronic case report form (eCRF) and in the source document. The documentation received from the IWRS indicating the code break must be retained with the participant’s source documents in a secure manner.

Participants who have had their study intervention assignment unblinded should continue to receive scheduled evaluations but will be excluded from primary analysis.

Intervention

Investigational drug: Patients in this study will be prescribed any formulation of tacrolimus authorised and commercialised in Spain.

1. Control arm: Will not receive any intervention based on their genetic profile; instead, they will receive the Standard of Care (SoC) tacrolimus dosing as determined by their healthcare provider.

Intervention arm: The tacrolimus dose will be prescribed according to the most recent clinical pharmacogenetic guideline treatment/dosing recommendations for their genetic profile.

Outcomes

Primary endpoint

Tacrolimus concentration levels on day 4 (±1 day) will be the effectiveness surrogate outcome. Number and percentage of patients achieving tacrolimus target plasma concentrations at visit 2. It will be considered in therapeutic range levels between 7 and 10 ng/mL.

Secondary endpoints

The following will be the study’s secondary endpoints:

• Incremental cost-effectiveness ratio (ICER): Ratio that divides the differences in costs between both treatments by the difference in effectiveness between both treatments.

• Number and percentage of patients with transplant rejection. Transplant rejection will be considered if there is histological confirmation and/or the patient initiates any type of therapy aimed at treating rejection (eg, corticosteroids).

• Rate of adverse events associated with treatment.

• Number and percentage of patients achieving tacrolimus target plasma concentrations at visits 4, 5 and 6.

• Healthcare expenditure related to predefined events of interest: Any costs made as a result of an adverse event.

Incidence of discontinuation or treatment modification due to lack of effectiveness related to the drug of inclusion.

Exploratory endpoints

The study includes two exploratory endpoints:

• Identification of new actionable genes/relevant polymorphisms within the predefined population subsets.

Novel prognostic and predictive genetic biomarkers of tacrolimus safety and effectiveness will be assessed using techniques that are not readily available at all centres but are accessible at a central laboratory affiliated with the Precision Medicine Infrastructure associated with Science and Technology (IMPaCT), as well as through genome-wide association studies (GWAS), when applicable. Given the limited sample size of the trial, these exploratory analyses will be interpreted with caution and considered only as hypothesis-generating for future research. IMPaCT is a national Spanish initiative aimed at advancing personalised healthcare. It is composed of three distinct pillars focused on genomics, population cohorts and biomedical data integration. Together, they are oriented towards improving R&D quality in the field of precision medicine. iPHARMGx is aligned with both the Genomic and Data pillars of IMPaCT.

Biological samples

After checking the selection criteria, informing the patient and obtaining the signed ICF, a sample for pharmacogenetic analysis will be collected. The pharmacogenetic sample should preferably be collected during the screening visit, but it can be collected at any point prior to the initiation of tacrolimus therapy. An aliquot of the DNA sample will be stored at −20°C for quality control purposes and/or further genetic analysis by techniques available at a central laboratory affiliated with the IMPaCT.

The subject must have a mandatory screening laboratory analysis, including all safety parameters described in this study, that can be performed between a window of 30 to 1 day before the baseline/randomisation visit (see Section 14 of TRANSPGx Study Protocol, (online supplemental material 2). Screening laboratory analysis may be conducted for any reason, as long as they fall within the aforementioned window and can in theory be dated before the signing of the ICF.

In alignment with the principles outlined in the iPHARMGx master protocol, our study will employ a proven pharmacogenetic genotyping tool. All centres participating in this study will genotype high-evidence (level 1A) relevant variants of the pertaining genes (table 2) according to their own available techniques. In this regard, and considering that any genotyping tool capable of interrogating the mandatory single nucleotide variants (SNVs) for this trial will be considered acceptable as per protocol, the preferred method for genotyping will be TaqMan probes. These can be used either individually or included in an OpenArray System that simultaneously analyses 120 SNVs. The turnaround time for results may vary depending on the laboratory, but it is expected to be no longer than 15 days from the time of sample collection. All participating local laboratories have reserved rights to analyse the aforementioned SNPs of a subject’s sample more than once, if they so wish to do so, for quality control purposes. However, they must ensure sufficient sample volume is preserved for central laboratory analysis. This will not be mandatory for all participating laboratories.

Table 2. Variants deemed mandatory to interrogate in all recruited subjects.

Gene	rsID	Star allele	cDNA
CYP3A5	rs776746	*3	6986A>G
CYP3A5	rs10264272	*6	14 690G>A
CYP3A5	rs41303343	*7	27131_27132insT

In those subjects that constitute outliers to previously established pharmacogenetic profiles, novel biomarkers will be explored through state-of-the-art techniques as described by the master protocol: genotyping will be performed at a central laboratory using advanced sequencing techniques with the aim of conducting an unbiased approach to potential biomarkers that may be associated with tacrolimus effectiveness and safety. Additionally, a sample from every genotyped subject will be stored as an aliquot and preserved to serve as a quality control measure. These aliquots can be made available for analysis at said central laboratory at any given time, to be explored through any technique deemed adequate by them.

Statistical analysis

A descriptive-univariate analysis will be carried out for all study variables. Frequency results will be expressed as absolute and relative frequencies. Continuous variables will be expressed with the main measures of dispersion (mean, SD, median and IQR). The normality of the variables will be studied using the Kolmogorov-Smirnov normality test.

For the study of the main variable, the Pearson Chi-Square test (or Fisher’s exact test for 2X2 tables or likelihood ratio in mXn tables, if necessary) will be used in the case of qualitative variables and the Student’s t-test, ANOVA of one factor or its non-parametric equivalents Mann-Whitney U test or Kruskal-Wallis test in the case of quantitative variables.

Finally, a multivariate model will be fitted with all the variables that were statistically significant in the previous analysis. Different multivariate models will be fitted (logistic regression, unweighted support vector machine, weighted support vector machine, artificial neural networks and partial least square regression). The optimal model will be selected using the Bayesian information criterion (BIC). The statistical software R [R Core Team (2014). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria] will be used.

The level of statistical significance is set at p<0.05. The statistical software R (version 4.3.1, 2023–06-16) [R Core Team (2021)] will be used.

Further details will be included in the Statistical Analysis Plan (SAP).

Economic evaluation

A cost-effectiveness evaluation will be conducted from the perspective of the Spanish National Healthcare System. Direct costs for each arm of the study will be calculated.

Direct costs will encompass healthcare resources used during the follow-up period, along with their associated unit costs per patient. Additionally, the expenses incurred by the participating centres to implement the intervention will be accounted for.

Various resources and their corresponding unit costs, including doctor’s visits, medications, and any hospital interventions during follow-up, will be recorded. The total costs for each patient in both intervention groups will be aggregated to allow for a comparison of the average cost per event avoided between the intervention and control groups. The findings will be presented in terms of cost per event and avoided events, with efforts made to assess the utility of each intervention whenever feasible.

To gauge the uncertainty surrounding the model and unit costs, particularly if obtained from sources other than the clinical centres, both univariate and probabilistic sensitivity analyses will be conducted. These analyses will provide valuable insights into the robustness of the findings and assist in decision-making processes.

Data collection and outcome measures

The handling, communication and transfer of personal data will be protected, complying with the basic ethical principles of biomedical research and applicable regulations: Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data and repealing Directive 95/46/EC (General Data Protection Regulation) and Act 41/2002 of 14 November, regulating patient autonomy and rights and obligations regarding clinical information and documentation. An eCRF form has been designed using MACRO Electronic Data Capture by Elsevier. To ensure the quality of data, data management will be performed by a data manager of the Spanish Clinical Research Network (SCReN), located at University Hospital La Paz. The data management plan has been approved by the principal investigator. Data collection forms will be included in the final report. All demographic data (including the subject’s gender, age and ethnicity) will be recorded in the eCRF and factored in the analysis plan. On completion of the study, all participants will be informed of their pharmacogenetic profile, regardless of the study arm to which they were allocated. Adverse drug events (ADEs) will be graded according to the Common Terminology Criteria for Adverse Events (CTCAE) V. 5.0 and are recorded in the electronic eCRF for both reporting and statistical analyses according to the hierarchical structure of the Medical Dictionary for Regulatory Activities (MedDRA), categorising events under the relevant system organ class (SOC) and, whenever possible, the MedDRA Preferred Term (PT).

Coordination, data management, monitoring and statistical analysis of the study will be performed by SCReN.

Patient and public involvement

The development of the research question and outcome measures was based on the clinical experience of nephrology specialists that treat this profile of patients and have an active desire to optimise tacrolimus treatment. Patients and patient advisers were not involved in the design, recruitment or conduct of this study. Patients will be informed of the study results both in writing and verbally and will be invited to contribute to the development of our dissemination strategy.

Ethics and dissemination

This study follows the principles of the 2013 Declaration of Helsinki. It was designed and shall be implemented, executed and reported in accordance with the ICH Harmonised Tripartite Guidelines for Good Clinical Practice, with applicable local regulations on clinical studies and patient data confidentiality. The study was reviewed and approved by the Spanish Regulatory Agency (AEMPS) and by the Clinical Research Ethics Committee of La Paz University Hospital (HULP code: 6845). The researchers will adhere strictly to the provisions of this protocol and will complete the case report forms. The investigators ascertain they will apply due diligence to avoid protocol deviations. If deviations occur, the investigator must inform the monitor, and the consequences of such deviations will be reviewed and discussed by the team. All protocol deviations will be documented/recorded specifying the reason, date, action taken and consequences to patients and the study.

Any protocol amendment will be identified by a consecutive number and must be approved and signed by the sponsor and the principal investigator. If relevant, approval by the Ethics Committee and the Spanish Agency of Medication and Health Products must occur before any changes are implemented.

This clinical trial has been classified by the Spanish Agency for Medicines and Healthcare Products as a ‘low-intervention clinical trial’. The additional diagnostic or monitoring procedures do not pose more than minimal additional risk or burden to the safety of the subjects compared with normal clinical practice. If there is any possible damage that could be suffered by a subject, resulting from the use of the investigational medicinal product in accordance with the protocol of that specific clinical trial, it is covered by the applicable compensation system already in place.

The investigators ascertain they will apply due diligence to avoid protocol deviations. If deviations occur, the investigator must inform the monitor, and the consequences of such deviations will be reviewed and discussed by the team. All protocol deviations will be documented/recorded specifying the reason, data, action taken and consequences to patients and the study.

Anonymised individual participant data will be made available when the trial is complete, on request to the corresponding authors and approval by the scientific committee. Once a proposal has been approved, data will be shared through a secure online platform. In addition, after study completion and finalisation of the study report, the results of this trial will be submitted for publication in a scientific journal or will be made public, complying with the Declaration of Helsinki.

Access to data

A copy of the database of data collected during the trial will be attached as an appendix to the publication resulting from this clinical trial. Data will be available at the same time as the results are published and will be kept available to everyone without any time limit. Data will be available indefinitely on the publisher’s website, as long as it is kept by the publisher, for anyone who wishes to access the data for non-commercial purposes.

Discussion

Several hurdles are precluding the seamless integration of pharmacogenetic biomarkers into patient care pathways. Challenges such as the scarcity of high-level evidence regarding the efficacy, effectiveness and efficiency of genetic markers, as well as a lack of consensus in the field on whether a widespread adoption would be feasible. The iPHARMGx Consortium’s pioneering initiative addresses critical barriers to the widespread implementation of pharmacogenetics in routine clinical practice.

The development of the TRANSPGx study would help optimise the treatment of patients receiving a renal transplant by aiming to achieve target plasma concentrations and reduce the impact of adverse events, thereby improving both patients’ quality of life as well as the overall sustainability of the Spanish National Health System. Patients with prior CYP3A5 genotyping are excluded in this trial to simulate a fully pre-emptive PGx strategy. This approach strengthens the internal validity, although it may limit generalisability in settings where genotyping is already being performed in clinical practice.

The establishment of comprehensive pharmacoeconomic evidence serves a dual purpose. First, it contributes to the expanding body of evidence that advocates for the viability of a personalised medicine strategy in tacrolimus management, thereby facilitating its broader adoption within the healthcare system. Second, it will provide in vivo evidence of the influence of various genotypes on tacrolimus effectiveness beyond the current research landscape. Simultaneously, it will offer insight into uncovering novel biomarkers that could help explain the often-encountered outliers in routine clinical practice, deepening our knowledge of immunosuppressor pharmacogenetics as a whole.

As of 17 January 2025, this study has received approval from an ethics committee and the AEMPS. The study was initiated on 31 March 2025, with the recruitment of the first subject on 3 June 2025. As of 30 June 2025, a total of three patients have been enrolled in the study. Recruitment and inclusion of the last subject are expected in December 2026, with a predicted end of follow-up in September 2027.