Immunosupprresive Therapy

#### **Chapter 9**

## Induction Therapy in the Current Immunosuppressive Therapy

*Takuya Watanabe, Yasumasa Tsukamoto, Hiroki Mochizuki, Masaya Shimojima, Tasuku Hada, Satsuki Fukushima, Tomoyuki Fujita and Osamu Seguchi*

#### **Abstract**

The current immunosuppressive therapy including calcineurin inhibitors, mycophenolate mofetil, and steroids, has substantially suppress rejections and improved clinical outcomes in heart transplant (HTx) recipients. Nevertheless, the management of drug-related nephrotoxicity, fatal acute cellular rejection (ACR), antibody-mediated rejection and infections remains challenging. Although previous some studies suggested that perioperative induction immunosuppressive therapy may be effective for the suppressing ACR and deterioration of renal function, increased incidence of infection and malignancy was concerned in recipients with induction immunosuppressive therapy. The international society of heart and lung transplantation (ISHLT) guidelines for the care of heart transplant recipients do not recommend routine use of induction immunosuppressive therapy, except for the patients with high risk of acute rejection or renal dysfunction, however, appropriate therapeutic regimen and indication of induction immunosuppressive therapy remains unclear in HTx recipients. We review current evidence of induction immunosuppressive therapy in HTx recipients, and discuss the appropriate therapeutic regimen and indication of induction therapy.

**Keywords:** induction therapy, interleukin-2 receptor antagonists, polyclonal anti-thymocyte antibodies, acute cellular rejection, renal dysfunction

#### **1. Introduction**

Triple immunosuppressive therapy including calcineurin inhibitors (CNI), anti-metabolites, and steroids, has substantially improved clinical outcomes for heart transplant (HTx) recipients. Nevertheless, the management of CNI-related nephrotoxicity, fatal acute cellular rejection (ACR), antibody-mediated rejection (AMR), and infections remains challenging [1]. Immunosuppressive regimens for organ transplantation can be generally characterized as induction, maintenance, or rescue therapies [2]. Recently, desensitization therapy has also been considered for recipients who are highly sensitized to Human leukocyte antigen (HLA) or have donor specific HLA antibodies [3]. Induction immunosuppressive therapy is a powerful and prophylactic therapy that is used perioperatively to prevent episodes of acute rejection, which is expected to improve the clinical prognosis or make their managements easier in high-risk HTx recipients. Currently, approximate 50% of HTx recipients employ a strategy of induction therapy, however, international clinical guidelines do not recommend the routine use of induction immunosuppressive therapy since the impact of induction therapy on survival in HTx recipients remains unclear [1]. In the more recent clinical situation, tacrolimus, which is recent alternative choice of cyclosporine, significantly reduces the incidence of ACR. And desensitization therapy is also becoming an established medical treatment for sensitized HTx recipients. Appropriate indications and therapeutic regimens for administering induction immunosuppressive therapy to HTx recipients requires further consideration in the recent clinical situations.

This manuscript will provide an overview of the induction immunosuppressive therapy up to now, and future perspective of the induction immunosuppressive therapy in the new era of the current more established immunosuppression.

#### **2. Induction immunosuppressive therapy in HTx**

#### **2.1 Immune response system in transplant recipients**

Immune response system that influences the rejection in transplant recipients is divided into two categories depending on the immune cells that primarily work, although each response influences the other; T-cell-mediated and antibody-mediated immune response.

#### *2.1.1 T-cell mediated immune response*

T-cell mediated immune response system in transplanted recipients is generally explained from three pathway; direct and semi-direct pathway which donor antigen presentation cell (APC) affect, and indirect pathway which recipient APC (**Figure 1**) [2]. Thymic selection in the native thymus occurs without regard for donor-specific allo-antigens. The naïve T cell has a relatively high allo-specific precursor frequency (**Precursor frequency**). This process can be nonspecifically reduced by depletion induction immunosuppressive agents including anti-thymocyte antibodies (ATG), muromonab-CD3 (OKT3), and alemtuzumab (**Figure 1a**). Allo-antigen is presented via donor (direct or semi-direct) or recipient-itself (indirect) APCs in the secondary lymphoid tissues inducing naïve T cell activation (**Antigen presentation**). In transplantation, graft derived APCs likely dominate this process early through reperfusion induced mobilization to the secondary lymphoid tissue and direct pathway. This pathway gives way to recipient derived migratory APCs later through indirect mechanisms and may also be influenced by semi-direct presentation of intact donor HLA by recipient cells. T-cell depleting agents, Interleukin 2 receptor (IL2R) blockage, and methylprednisolone limit this process (**Figure 1b**). T-cell activation occurs as an aggregate effect of many spectral processes (**Activation threshold**). Given that T cells have long been known to be important in rejection, some maintenance immunosuppressive agents including CNI, anti-metabolites and mammalian target of rapamycin (mTOR) inhibitors also alter the threshold of activation of T-cell also affect this process (**Figure 1b** and **c**). T-cells activation in the secondary lymphoid and injured endothelium and ischemic injury (**Figure 1A**) attenuates platelet and

*Induction Therapy in the Current Immunosuppressive Therapy DOI: http://dx.doi.org/10.5772/intechopen.103746*

**Figure 1.** *T-cell mediated immune response.*

complement binding and activation thus activating endothelial cells and donor APCs, initiating chemotactic signals, and providing signals to lower the activation threshold of local effector cells (**Figure 1B**). The local cytokine milieu reinforces local cell activation and can be inhibited by IL2R-specific agents, methylprednisolone, CNIs and mTOR inhibitors (**Figure 1d**). Allo-sensitized memory cells and cells activated through heterologous immunity or homeostatic proliferation bypass the need for nodal presentation. Depletion agents can both attenuate and augment this effect (**Figure 1e**). Activated T cells and recipient APCs are attracted to the graft site by chemokines and adhesion molecule expression (**Cell Trafficking**). Reperfusion injury initiates donor derived APCs to mobilize toward the nodes for direct pathway. Depletion agents, polyclonal antibody and methylprednisolone limit chemotaxis and/ or adhesion. Cytotoxic T lymphocyte (CTL) encounter the graft in sufficient numbers to cause clinical damage, and are reenforced by a milieu rich in T cell derived cytokines (e.g. IL-2) (**Effector response**). Damage to the organ occurs through contact dependent CTL activity and through the direct effect of cytolytic cytokines (e.g. TNF-α). Depletion agents and selective IL-2 receptor antibodies limits the productiveness of this response and prevents the attainment of milieu that is supportive of CTL activity (**Figure 1e**).

#### *2.1.2 Antibody-mediated immune response*

Anti-body mediated rejection (AMR) is a major limitation to long-term HTx survival and is mainly driven by antibodies directed against the mismatched HLA Class I and Class II antigens (HLA antibodies) expressed on the allograft. Pre-sensitized patients who possess HLA antibodies are disadvantaged by having to wait longer to receive an organ from suitably matched donor. The number of pre-sensitized patients has been increasing, a trend that is likely due to the increased use of mechanical circulatory assist devices [4]. The humoral immune system is responsible for antibody production, which leads to AMR (**Figure 2**) [5]. Naïve B-cells are produced in the bone marrow and become activated in secondary lymphoid tissues when antigen

*Antibody-mediated immune response.*

is encountered in the presence of APC and T-helper cells. Activated B-cells develop either into plasma blast secreting low-affinity antibody or interact with follicular dendritic and T-helper cells to form germinal centers [6]. Within germinal centers, B-cells undergo proliferation, hypermutation and affinity maturation to become high-affinity antibody-secreting plasma cells or memory B-cells. Plasma cells migrate back to the bone marrow, whereas memory B-cells circulate through secondary lymphoid organs and in the peripheral circulation. Upon re-exposure to antigen, memory B-cells rapidly proliferate and differentiate into plasma cells, producing high-affinity class-switched antibodies. Sensitized patients, who have already donor-specific antibodies pre-transplantation or memory B-cells against donor HLA by previous exposure, have high risk of hyperacute humoral rejection after HTx. In addition, antibody-mediated allograft injury occurs through complement pathway activation. HLA antibody-antigen complexes on allograft endothelial cells activate C1 triggering complement cascade activation and formation of the C5b-9 membrane attack complex to cause endothelial-cell lysis and destruction. Complement products also cause injury through recruitment of inflammatory cells (C3a, C4a, C5a), mastcell histamine release (C5a), upregulation of endothelial adhesion molecules (C5a), tissue factor synthesis and thrombotic injury (C5a, C5b-9) and Weibel-Palade bodies (WPB) exocytosis [7]. DSA also exert harmful effects independent of complement activation through Fc-receptor recruitment of inflammatory cells and release of inflammatory mediators. The resulting cellular inflammation, thrombosis, hemorrhage and lysis cause allograft injury and dysfunction.

Desensitization therapy is a specific and important option for increasing donor pool and access to transplantation for the sensitized patient, which reduces or eliminates HLA antibody and/or facilitates transplantation in the presence of DSA. Since T-B-cell interaction is also associated with the plsma-cell antibody production, T-cell directed therapy including mycophenolate acid is also considered as a desensitization therapy. ATG, an option for induction therapy, binds to cell surface antigens on T cells to injure and reduce T cells. Since humoral immune responses are suppressed

when helper T cell function is reduced, ATG has the effect of decreasing sensitization by suppressing T-B cell interactions. Other agents specific to desensitization do not necessarily suppress the T dell mediated immune response. Previous consensus report suggests that post-transplant induction therapy as well as standard maintenance immunosuppression is recommended to prevent rejection in patients who have undergone desensitization [8].

#### **2.2 Induction therapy in the current clinical situation**

Historically, all organ transplantation employed induction regimens using some immunosuppressive agents [2]. Their strategies include preoperative high dose therapy with maintenance drugs, including glucocorticosteroids, antimetabolites and intravenous CNI, or specialized induction agents such as antibodies or infusion proteins. The concept that more immunosuppression is required early after transplantation is well established regarding induction therapies to prevent rejections. Specialized induction immunosuppressive agents which do not affect worsening renal function are used in the early perioperative management of patients with known or worsening renal insufficiency, as it may enable delayed initiation with calcineurin inhibitors to prevent the development of acute renal failure. Major concerns of induction therapy may be increased risk of infection and malignancy. Specialized induction immunosuppressive agents can largely be divided into two categories: depleting antibodies and non-depleting antibodies [2]. Depleting antibodies include both monoclonal (OKT3 and alemtuzumab) and polyclonal (ATG) antibodies. Depleting antibodies reduce alloreactive T cells at the time of transplantation, in turn suppressing host response to the allograft. As depleting antibodies acts primitive T-cell and also indirectly suppresses the anti-body mediated response via B-cell, resulting in a stronger suppression of immune responses more than non-depleting antibodies. While, as nondepleting antibodies inhibit T-cell activities which acts against a downstream of immune-response cascade (such as IL-2-driven cell proliferation), it may suppress rejections more specifically.

#### *2.2.1 Current trend of Induction therapy regimens*

Cai and Terasaki reviewed renal transplant recipients in the United Network for Organ Sharing (UNOS) database, [9] there had been three distinct time periods of induction regimens: (1) 1987–1993, the old, low-induction antibody era, when fewer than 30% of all kidney recipients received induction therapy, consisting mostly (80%) of anti-lymphocyte globulin or OKT3; (2) 1994–2002, the transitional, high-induction antibody era, when approximately 80% of kidney transplant recipients received induction therapy, and anti-lymphocyte globulin and OKT3 starting to be replaced by daclizumab (1998), basiliximab (1998), and rATG (1999); and (3) 2003–2010, the modern high-induction antibody era, with induction therapy remaining high, more than 80% of all transplant patients receiving induction therapy, mostly rATG, basiliximab, daclizumab, or alemtuzumab (2003). Regarding to HTx recipients, Whitson et al. evaluated the usefulness of induction therapy using UNOS database from 2001 to 2012 in HTx recipients [10]. Of the 17,857 HTx recipients, 8216 (46%) recipients had induction therapy; 55% were IL-2R antibodies (IL-2RA), 40% some depletion agents including ATG, and 4% alemtuzumab. Nozohoor et al., reviewed 27,369 adult HTx recipients in the International Society for Heart and Lung Transplantation (ISHLT) registry database, showed that 11,681 (43%) recipients had

induction therapy; 59% were ATG and 41% basiliximab [11]. Tzani et al. showed the trend in induction therapy utilization in patients who underwent HTx from 1990 to 2020, using UNOS Registry Standard Analysis and Research database [12]. The utilization of induction therapy gradually increased, reaching almost 50% in 2006, and then maintained similarly until 2016, with a recent gradual decrease to almost 40 % of all HTx in 2020. The use of alemtuzumab and OKT3 decreased significantly while the use of IL-2RA and ATG increased, and since 2003, IL-2RA has been used primarily as induction therapy. The international registry data base has also showed that almost 50% of HTx programs employ a strategy of induction therapy. Although multitude induction agents are available as mentioned above, IL-2RA and polyclonal ATG were commonly used [1].

#### *2.2.2 Current clinical implication of induction therapy*

The purpose of induction therapy is primarily to achieve high intensity immunosuppression early in the postoperative period to reduce the incidence of rejection and to delay the initiation of nephrotoxic immunosuppression with CNI in recipients with compromised renal function [9]. In addition, reduced risk of incidence of rejection may result in suppressing the development of cardiac allograft vasculopathy [13]. The potential disadvantage of induction therapy is the increased risk of infection in early phase and malignancy in the long-term post-HTx [13]. A previous meta-analysis showed that acute rejection might be reduced by induction therapy compared with no induction, and did not show other clear survival benefits or harms associated with the use of any kind of T-cell antibody induction agents compared with no induction [14]. Another systematic review showed that patients receiving induction therapy had similar risk of moderate-to-severe rejection, all-cause death, infection, and cancer with patients who did not receive induction therapy [15]. A more recent retrospective analysis using large cohort date of UNOS registry showed that induction therapy was associated with lower mortality and treated rejection episodes than no induction therapy [12].

In the current clinical situation, the improvement and establishment of new maintenance immunosuppression agents such as tacrolimus replaced cyclosporine and mycophenolate mofetil replaced azathioprine have significantly reduced risk of acute T-cell mediated rejection in acute phase post-HTx, which may lead that previously observed benefits of induction therapy tend to decrease overtime. Thus, although the clinical need of induction therapy to suppress T-cell mediated rejection may be decreasing, younger patients, multiparous women, African Americans, patients with longer term ventricular assist device, [16] and patients with long ischemic time [17] may be still good indication for the induction therapy in HTx. On the other hand, long awaiting time for HTx due to the severe donor shortage and increasing in the implantation of left ventricular assist device pre-HTx have increased risk of sensitization and pre-existing renal dysfunction before HTx. Highly sensitized patients, and those with positive cross-match may also have been considered as the candidate for the induction therapy in the past, however, since evidence for desensitization therapy is being established, truly high risk patients for hyperacute antibody-mediated rejection with high intensity of donor-specific should be considered more specific desensitization rather than introduction immunosuppressive therapy. And induction therapy may be generally used in combination with desensitization therapy, not induction therapy alone [3, 5]. Patients with pre-existing renal dysfunction may still be the best indication of induction therapy in the current clinical situation [17–20].

#### **2.3 Specific agents for induction therapy**

There are many specialized induction agents that are now being used to target the components of immunity heightened during transplantation. Although there is positive evidence in randomized trials and prospective studies comparing with standard maintenance regimens, no-induction or methylprednisolone induction, most trials use the surrogate endpoint of acute rejection, rather than more definitive outcome measures such as patient or graft survival. Several induction regimens have shown to measurably increase the risk of posttransplant lymphoproliferative disease (PTLD) and death from malignancy when combined with conventional maintenance immunosuppression [21]. This manuscript focuses on two specific induction immunosuppressive agents which were commonly used in current clinical situations; ATG and IL-2RA.

#### *2.3.1 Polyclonal antibody*

ATG is a polyclonal antibody derived from immunization of mainly rabbits with human thymocytes. The final product includes antibodies against multiple cell surface proteins, and HLA class 1 heavy chains, and is effective in preventing cellular immune responses against a variety of antigenic stimuli, through substantial lymphocyte depletion. Namely, ATGs bind to several antigens on T- and B-cells, causing T- and B-lymphocyte depletion. Given their broad spectrum of specificity, they have frequently been suggested to mediate their anti-rejection properties through means other than depletion, including costimulation blockade, adhesion molecule modulation, and B cell depletion. ATG is the most commonly used induction agent. Around 20% of HTx recipients receive ATG as induction therapy. There are no studies comparing ATG induction therapy with no induction therapy [15], and the efficacy of ATG induction therapy has been investigated in comparison with induction therapy with IL-2RAs which already showed the significant reduction of rejections. A large multicenter study has observed lower rates of rejection and an increased risk of infection with ATG [22].

The xenogeneic (horse or rabbit) origin of ATG may induce a host antibody response leading to acute hypersensitivity response or rarely, serum sickness on subsequent exposure, which is characterized by fevers, chills, tachycardia, hypertension or hypotension, myalgias, and rash, and may occur after the first dose. Rarely, cytokine release syndrome can occur. Furthermore, these ATGs cannot be used repeatedly for rejection to avoid a second or subsequent allergic reaction. ATG mat be left aside for future refractory rejections, not using for introduction.

#### *2.3.2 Interleukin 2 receptor antibody*

The high affinity alpha chain IL2 receptor (CD25) was the first molecule to be successfully targeted with a humanized monoclonal antibody in solid organ transplantation. IL-2RA act through the binding of the IL-2 receptor located on activated T-cells, thereby inhibiting the proliferation and differentiation of T-lymphocytes. Basiliximab is a monoclonal antibody that selectively binds to the IL-2 receptor of T-lymphocytes, blocks binding of IL-2 to the receptor complex, and inhibits IL-2 mediated T-lymphocyte proliferation [23]. Daclizumab is a humanized anti-IL-2R (CD25) monoclonal antibody that has the murine antigen-binding sequences molecularly engrafted onto a human antibody [24]; however, daclizumab has since been

discontinued by the manufacturer due to diminishing use. Basiliximab is notable for a significantly lower incidence in drug-related adverse events [25], compared with other specialized agents for induction therapy. Cytokine release syndrome has not been reported after administration of this type of drug.

Three randomized trials have compared with IL-2RA vs. no induction [23, 24, 26]. A systematic review including these randomized trials showed that IL-2RAs significantly reduced the risk of acute rejection. However, because these randomized trials had a high risk of bias despite randomization, this significant superiority of the IL-2 receptor was not clear according to the random effects model. Its survival benefits were also not found [27]. Furthermore, most of the studies to date have been in HTx recipients who received cyclosporine rather than tacrolimus for primary immunosuppression, with limited evidence in the new immunosuppression era. Watanabe et al. in HTx recipients receiving tacrolimus showed that basiliximab-based induction immunosuppressive therapy might suppress mild acute cellular rejection, and improve renal function in recipients with deteriorated renal function, and resulting in the its non-inferior outcome as compared to no-induction group even in recipients with any comorbidity [17].

#### *2.3.3 Current evidence of comparison ATG vs. IL-2 RA*

Although two randomized controlled trials demonstrated that the IL-2RA, daculizmab, effectively reduced the rate of moderate and severe rejections within first year after HTx [12, 23, 24], such effect could not be observed in trials for ATG. Previous systematic review which evaluated four randomized trials comparing of ATG with IL-2RA [28–31] showed that the use of IL-2RA was associated with significantly higher risk of moderate-to-severe rejection than ATG, but similar risk of death, infections, and malignancy [15]. In the retrospective analyses using large registry or cohort data in HTx, Nozohoor et al. [11] suggested that the recipients receiving ATG showed the better survival as compared with those receiving IL-2RA, however, found more malignancy post-HTx with ATG compared with basiliximab. Tzani et al. [12] showed that ATG has lower risk of treated rejection and mortality as compared with IL-2RA. And Ansari et al. in the retrospective analysis showed similar one-year survival between ATG and IL-2RA, but IL-2RA exhibited decreased long-term survival compared with ATG at 5 years and 10 years post-HTx [32]. On the other hand, Mazimba et al. [33] showed a conflict results when patients were stratified using risk of infection and rejection; IL-2RA was lower incidence of rejection but increased costs for infection in the patients with low risk of rejection and high risk of infection, and had significant lower incidence of rejection in patients with high risk of rejection and low risk infection as compared with ATG. A potential disadvantage of induction therapy is a risk of malignancies induced by its excessive immunosuppression in the long-term post-HTx [34]. ATG depletes cytotoxic T lymphocytes against organisms and virus infected cells as well as transplant organs. Therefore, ATG-based induction therapy may cytotoxic T lymphocytes against Epstein Barr virus (EBV) and EBV infected B lymphocytes which may result in primary-like EBV infection and EBV related B cell type posttransplant lymphoproliferative disorder (PTLD). Most previous studies did not show the difference of the incidence of malignancy between ATG- and IL-2RAbased induction therapies. Nozohoor et al. showed that the use of ATG may be associated with increased malignancy-related mortality, compared with no-induction [11]. Especially in pediatric HTx, ATG-based induction therapy tends to be preferred to IL-2RA-based induction therapy in younger patients, in those with congenital heart diseases, in patients requiring pre-transplant inotropic or mechanical support, and

in more sensitized patients or those with longer ischemic time [35]. Children are at greatly increased risk of PTLD versus adults, and PTLD is the most common form of post-transplant malignancy in children [36]. Although the relative rarity of PTLD makes an accurate assessment of the effect of specific immunosuppressive agents difficult, a recent review concluded no increased risk of PTLD in children given ATG after pediatric HTx [35]. They speculated that it is possible that this reduction in risk may have arisen from the general trend towards less intensive maintenance therapy in recent years. ATG-based induction may also have been used to facilitate CNI-sparing or steroid sparing therapy in pediatric HTx, potentially lowering risk the risk for PTLD.

Regarding maintenance immunosuppression, tacrolimus is more potent than cyclosprone and has proven to reduce rejection rates as well as an effective rescue agent for patients with recurrent or refractory acute allograft rejection. Tacrolimus has replaced cyclosporine in many transplant centers and currently. This raises the question about effectiveness of induction therapy in current tacrolimus-based immunosuppression era. Ali et al. performed meta-analysis to explore the effect of IL-2RA vs ATG on morbidity and mortality in renal transplant patients receiving tacrolimus-based maintenance immunosuppressive therapy, which revealed no significant difference in patient and graft survival when using IL-2RA vs ATG with the tacrolimus-based maintenance immunosuppression. The difference in efficacy between ATG and basiliximab in the era of newer immunosuppressive agents needs to be explored in HTx recipients.

ATG and IL-2RA may not be compared identically as induction therapy because the pharmacological mechanisms of action, response range, and safety of the two immunosuppressive agents are very different. Induction therapy with desensitization in highly sensitized patients or patients with donor specific antibodies may be not sufficient for basiliximab, and ATG should be selected as induction therapy. On the other hand, if induction therapy is administered because of concerns about worsening renal function immediately after transplantation in non-sensitized recipients, ATG may not be appropriate because it may lead to excessive immunosuppression, and the use of safer may be appropriate. Furthermore, since xenogeneic origin of ATG, ATGs cannot be used repeatedly for rejection to avoid a second or subsequent allergic reaction, ATG may require to be left aside for future refractory rejections.

#### **3. Future perspective regarding the induction therapy**

#### **3.1 Appropriate indication for induction therapy**

The appropriate indications for administering induction therapy have not been established. Previous studies suggested that recipients with an increased risk of rejection, which were younger patients, multiparous women, African Americans, patients with longer term ventricular assist device [16], and patients with long ischemic time [17], are good indication for the induction therapy in HTx, as well as recipients with deteriorated renal function. Watanabe et al. proposed the original indication criteria which included potential difficulty in patient management including donor or recipient older age, impairment of cardiac function or pre-existing coronary atherosclerosis of donor heart in early phase after HTx which may cause intolerance to immunosuppression.

#### **3.2 Appropriate regimens for induction therapy**

There is currently no consensus regarding the dose or duration of induction agents in different types of HTx recipients, or the timing and intensity of initial CNI therapy in recipients receiving induction therapy. The immunosuppression protocols for administering induction therapy varies according to the dosage of CNI administered and applies to those recipients who require CNI withdrawal with cytolytic therapy for renal dysfunction or as a modification of the standard triple immunosuppression regimen [23, 24, 27]. And these regimens influence perioperative over- or under immunosuppression particularly, and need to be careful in patients with administered induction therapy. Minimization and optimization of baseline immunosuppressive agents may be useful for improving clinical outcomes. Regarding the optimization of maintenance immunosuppression, some landmark trials in CNI minimization and withdrawal shows the clinical usefulness, however, perioperative optimization in immunosuppression in patients with induction therapy is still controversial [23, 24, 27]. When considering the optimal immunosuppressive regimen with induction therapy, it may be useful to monitor the degree of immunosuppression. Previous review paper suggested that CD3 monitoring, or absolute lymphocyte count is useful to guide ATG dosing [35]. Where this approach is applied, the previous ISHLT guideline advise targeting a CD3 count in the range of 25–50 cells/mm3 , or an absolute total lymphocyte count <100–200 cells/mm3 [37]. A previous small sample retrospective study showed the patient group managed with CD3 monitoring received a significantly lower total ATG dose, although clinical outcome including survival, rejection and infection did not differ [38]. Regarding IL-2RA based induction therapy, CD25 which expressed on activated T lymphocytes may be useful for assessing the effects of IL-2RA. A previous study monitoring the CD25 count to evaluate the effect of IL2-RA showed that a 2-dose regimen of basiliximab-based induction therapy administered on Day 0 and Day 4 after transplantation still suppressed T-lymphocyte activation for an average 40–50 days after renal transplantation [39]. Watanabe et al. performed an original regimen that CNI dosage was slowly increased to prevent further deterioration of renal dysfunction due to CNI-induced kidney injury for the recipients with renal dysfunction, and to prevent over-immunosuppression for the pretransplant sensitized recipients; trough level of tacrolimus in the induction group was significantly lower than that in the no-induction group until 3 weeks post-HTx. However, recipients receiving induction therapy showed significantly higher incidence of infectious disease. Further investigation is needed for appropriate regimens for induction therapy.

#### **4. Conclusions**

This manuscript reviews previous and more current evidence of induction therapy in HTx recipients, and discussed the appropriate therapeutic regimen and indication of induction therapy in the current clinical situation. In previous evidence, conflicting results have been reported with regard to the effect of induction therapy on longterm survival, also the comparison between ATG and IL-2RA. Appropriate patient selection and agent selection may maximize the efficacy of induction therapy. The proper use of induction therapy is still being determined. Recent advances in immunosuppressive agents have changed the clinical course of HTx recipients. Induction therapy should be selected, specifically based on their mechanism of action to specific clinical need and aim.

#### **Acknowledgements**

This work was supported by a Japan Heart Foundation Research Grant and by the JSPS KAKENHI [grant number JP19K09256] to T.W., and was supported by the JSPS KAKENHI [grant number 21H0921] to Yasunori Shintani.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Takuya Watanabe1 \*, Yasumasa Tsukamoto1 , Hiroki Mochizuki1 , Masaya Shimojima1 , Tasuku Hada1 , Satsuki Fukushima<sup>2</sup> , Tomoyuki Fujita<sup>2</sup> and Osamu Seguchi1

1 Department of Transplant Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan

2 Department of Cardiovascular Surgery, National Cerebral and Cardiovascular Center, Osaka, Japan

\*Address all correspondence to: watanabe.takuya@ncvc.go.jp

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[22] Emin A, Rogers CA, Thekkudan J, Bonser RS, Banner NR. Antithymocyte globulin induction therapy for adult heart transplantation: A UK national study. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2011;**30**:770-777

[23] Beniaminovitz A, Itescu S, Lietz K, Donovan M, Burke EM, et al. Prevention of rejection in cardiac transplantation by blockade of the interleukin-2 receptor with a monoclonal antibody. The New England Journal of Medicine. 2000;**342**:613-619

[24] Hershberger RE, Starling RC, Eisen HJ, Bergh CH, Kormos RL, et al. Daclizumab to prevent rejection after cardiac transplantation. The New England Journal of Medicine. 2005;**352**:2705-2713

[25] Nashan B, Moore R, Amlot P, Schmidt AG, Abeywickrama K, Soulillou JP. Randomised trial of basiliximab versus placebo for control of acute cellular rejection in renal allograft recipients. CHIB 201 International Study Group. Lancet (London, England). 1997;**350**:1193-1198

[26] Mehra MR, Zucker MJ, Wagoner L, Michler R, Boehmer J, et al. A multicenter, prospective, randomized, double-blind trial of basiliximab in heart transplantation. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2005;**24**:1297-1304

[27] Moller CH, Gustafsson F, Gluud C, Steinbruchel DA. Interleukin-2 receptor antagonists as induction therapy after heart transplantation: Systematic review with meta-analysis of randomized trials. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2008;**27**:835-842

[28] Bonaros N, Dunkler D, Kocher A, Imhof M, Grimm M, et al. Ten-year follow-up of a prospective, randomized trial of BT563/bb10 versus antithymocyte globulin as induction therapy after heart transplantation. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2006;**25**:1154-1163

[29] Carrier M, Leblanc MH, Perrault LP, White M, Doyle D, et al. Basiliximab and rabbit anti-thymocyte globulin for prophylaxis of acute rejection after heart transplantation: A noninferiority trial. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2007;**26**:258-263

[30] Mattei MF, Redonnet M, Gandjbakhch I, Bandini AM, Billes A, et al. Lower risk of infectious deaths in cardiac transplant patients receiving basiliximab versus anti-thymocyte globulin as induction therapy. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2007;**26**:693-699

[31] Mullen JC, Kuurstra EJ, Oreopoulos A, Bentley MJ, Wang S. A randomized controlled trial of daclizumab versus antithymocyte globulin induction for heart transplantation. Transplantation Research. 2014;**3**:14

[32] Ansari D, Lund LH, Stehlik J, Andersson B, Hoglund P, et al. Induction with anti-thymocyte globulin in heart transplantation is associated with better long-term survival compared with basiliximab. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2015;**34**:1283-1291

[33] Mazimba S, Tallaj JA, George JF, Kirklin JK, Brown RN, Pamboukian SV. Infection and rejection risk after cardiac transplantation with induction vs. no induction: A multi-institutional study. Clinical Transplantation. 2014;**28**:946-952

[34] Lindenfeld J, Miller GG, Shakar SF, Zolty R, Lowes BD, et al. Drug therapy in the heart transplant recipient: Part I: Cardiac rejection and immunosuppressive drugs. Circulation. 2004;**110**:3734-3740

[35] Schweiger M, Zuckermann A, Beiras-Fernandez A, Berchtolld-Herz M, Boeken U, et al. A review of induction with rabbit antithymocyte globulin in pediatric heart transplant

*Induction Therapy in the Current Immunosuppressive Therapy DOI: http://dx.doi.org/10.5772/intechopen.103746*

recipients. Annals of Transplantation. 2018;**23**:322-333

[36] Opelz G, Döhler B. Lymphomas after solid organ transplantation: A collaborative transplant study report. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2004;**4**:222-230

[37] Costanzo MR, Dipchand A, Starling R, Anderson A, Chan M, et al. The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 2010;**29**:914-956

[38] Thrush PT, Gossett JG, Costello JM, Matthews KL, Nubani R, et al. Role for immune monitoring to tailor induction prophylaxis in pediatric heart recipients. Pediatric Transplantation. 2014;**18**:79-86

[39] Haba T, Uchida K, Katayama A, Tominaga Y, Sato T, et al. Pharmacokinetics and pharmacodynamics of a chimeric interleukin-2 receptor monoclonal antibody, basiliximab, in renal transplantation: A comparison between Japanese and non-Japanese patients. Transplantation Proceedings. 2001;**33**:3174-3175

**Chapter 10**

## Role of the Transplant Pharmacist

*Megumi Ikura, Kazuki Nakagita, Takaya Uno, Hiromi Takenaka, Sachi Matsuda, Miho Yoshii, Rikako Nagata, Ichiro Nakakura, Naoki Hayakawa, Tsutomu Nakamura, Kyoichi Wada and Osamu Seguchi*

#### **Abstract**

At the National Cerebral and Cardiovascular Center, Japan, pharmacists have been involved in drug treatment management and patient care as members of multidisciplinary heart transplant teams that include surgeons, physicians, recipient transplant coordinators, and nurses during the waiting period for heart transplantation (HTx), HTx surgery, and post-HTx. During the waiting period, pharmacists play an important role in adjusting the use of antibiotics, anticoagulants, and antiarrhythmics by patients receiving a ventricular assist device (VAD). During HTx surgery and post-HTx, pharmacists advise physicians regarding the individualized medication protocol for immunosuppression and infection prevention to be used for each patient based on the patient's pre-HTx characteristics as well as gene polymorphisms. They thus contribute to reducing the burden on the physician through the sharing of tasks. Throughout all three phases of HTx, pharmacists repeatedly provide medication and adherence education to the patients and caregivers. It is hoped that an academic society-led training protocol as well as transplant pharmacists will be established in Japan and other developed countries, and that these specialized transplant pharmacists would then provide individualized pharmacotherapy for the use of various antibiotics, anticoagulants, and immunosuppressive agents that have a narrow range of treatment in VAD and HTx patients.

**Keywords:** transplant pharmacist, individualized therapy, patient education, immunosuppressive agents

#### **1. Introduction**

In 1997, the Act on Organ Transplantation was enacted in Japan, and the first heart transplantation (HTx) under the law was performed in 1999 [1]. However, the number of organ donors has been quite low in Japan, and therefore, many HTx candidates have no choice but to travel overseas to seek the opportunity for HTx [2]. Meanwhile, the Declaration of Istanbul was set out at the Transplantation Society in 2008, in which patients awaiting HTx need to wait for donor opportunities in their own countries, sidestepping efforts by many individuals related to organ transplantation to amend and revise the Act on Organ Transplantation in 2010. Over the last decade,

although the number of brain-dead organ donors has increased gradually, the number of patients awaiting HTx has been increasing year by year [2]. Furthermore, the COVID-19 pandemic reduced the frequency of the organ transplantation procedures, and the number of organ donors has decreased. Consequently, the waiting period for HTx has become longer, at 5 years or more in most cases in Japan [3].

At the National Cerebral and Cardiovascular Center (NCVC), Japan, the multidisciplinary HTx team, including surgeons, physicians, pharmacists, recipient transplant coordinators (RTC), nurses, nutritional support teams (NSTs), physical therapists, and medical engineers, has supported the patients during the waiting period for HTx, HTx surgery, and post-HTx. In this situation, pharmacists play the role of a specialist providing pharmaceutical care to patients awaiting HTx as well as heart transplant recipients (HTRs). The pharmacist stationed in the ward participates in morning and evening conferences as a member of the medical team to monitor the patient's daily condition, provides pharmacological management, and actively provides prescription support and patient education, and it is expected that the transplant pharmacist also actively contributes to individualized pharmacotherapy for various patient groups, from those suffering from severe heart failure to those in the post-transplantation phase.

In this chapter, the role and responsibilities of pharmacists are described from the perspective of drug treatment management and patient education in preoperative and postoperative HTx patients, and individualized pharmacotherapy is also discussed.

#### **2. Waiting for an HTx**

Patients awaiting HTx have terminal circulatory failure, and ventricular assist devices (VADs), which can mechanically propel blood from the heart to the central circulation and temporarily augment the cardiac output, have been recognized as essential treatment options as "bridge to transplant" (BTT) [1–3]. VADs include intracorporeal or paracorporeal devices, and the former can not only improve the functional status and quality of life of the patients awaiting HTx, but also enable them to return to almost normal lives [4, 5].

On the other hand, VADs often cause pump thrombosis by forming blood clots in the device, and therefore patients receiving VADs require long-term anticoagulation treatment to prevent thromboembolic complications. In addition, although intracorporeal VADs are fully implantable pumps in the body, a driveline attached to the pump penetrates the skin and connects to an external controller and battery, thereby potentially increasing the risk of infectious diseases that may require hospitalization.

At NCVC, intracorporeal VADs have been used in more than 90% of the patients awaiting HTx. The pharmaceutical management and patient education by pharmacists are described below.

#### **2.1 Pharmaceutical management**

#### *2.1.1 Warfarin (WF)*

Warfarin (WF) is most frequently used as a prophylactic antithrombotic drug after VAD implantation [6]. In general, the dose of WF is routinely adjusted according to the prothrombin time international normalized ratio (PT-INR). At the start of an urgent anticoagulant therapy for VAD implantation, heparin or dalteparin is used in combination

#### *Role of the Transplant Pharmacist DOI: http://dx.doi.org/10.5772/intechopen.102372*

with WF while paying attention to heparin-induced thrombocytopenia until stable PT-INR can be maintained. Meanwhile, antimicrobial agents such as linezolid (LZD), daptomycin (DAP), and levofloxacin, and antiarrhythmic agents such as amiodarone (AMD), may result in drug–drug interactions with WF, which could lead to unexpected anticoagulation and bleeding risk [7–9]. Pharmacists check classical clinical factors (age, sex, weight, height, and concomitant medication) and request the physicians in charge to take blood samples for additional PT-INR monitoring and provide prescription support, if a potential drug interaction between WF and concomitant medication is a concern.

WF produces an anticoagulant effect by interfering with the interconversion of vitamin K (VK) to its reduced form, which is required for γ-carboxylation of several vitamin-K-dependent proteins that regulate blood coagulation [6]. VK is present in many kinds of foods and beverages, and it has been reported that meals affect PT-INR in patients taking WF [7–9]. Therefore, careful attention should be paid to foods and drinks for the control of PT-INR. On the other hand, VK plays an important role in bone formation through activation of osteocalcin as well as maintenance of normal blood coagulation [10, 11], and excessive restriction of VK intake may lead to decreased quality of life. At NCVC, an interdisciplinary NST, composed of physicians, dieticians, pharmacists, and nurses, participates in the routine assessment of the patient's energy, protein, fluid, mineral, and electrolyte requirements as well as vitamins, and pharmacists explain the importance of dietary management to obtain stable PT-INR to the patients receiving VAD and their families/relatives during hospitalization [12]. A certified dietician also controls every HTR's VK intake through the meals not only during hospitalization but also after release from the hospital.

Recently, genetic polymorphisms of genes encoding cytochrome (CYP) 2C9, a metabolic enzyme of *S*-WF, and those of the VK epoxide reductase complex (VKORC1), a target enzyme of WF in vitamin K recycling, have also been reported as key factors affecting the pharmacokinetics (PK) and pharmacodynamics (PD) of WF, respectively. These factors may be useful for the control of PT-INR through dose adjustments [10, 11]. At NCVC, we often observe that some patients with an implanted VAD have difficulties in controlling the dose of WF. In such a case, pharmacists suggest the physician additional tests to determine the genotypes of CYP2C9 and VKORC with the consent of the patient and carefully adjust the WF dosage [11]. Because dose adjustments based on gene polymorphisms are not covered by the universal health insurance system in Japan so far, further evidence-based data accumulation is needed.

Meanwhile, when the patient with VAD undergoes urgent surgery or experiences severe or life-threatening bleeding, the dose of WF should be reduced immediately, and anticoagulation reversal is required. The International Society for Heart and Lung Transplantation (ISHLT) guidelines recommend that anticoagulation therapy should be held in patients with mechanical circulatory support in the setting of clinically significant bleeding [13]. In such emergency cases, in addition to the cessation of WF treatment, intravenous administration of vitamin K2 as an antagonist of the anticoagulant activity of WF and that of human prothrombin complex supplemented with VK-dependent blood coagulation factors are performed to improve the excessively enhanced anticoagulant state.

#### *2.1.2 Antimicrobial agents*

During the waiting period for HTx, VAD-associated infections can often be a problem. Pharmacists help physicians select appropriate antimicrobial agents and maintain their dosing adjustments. In addition, pharmacists routinely monitor side effects and sometimes utilize therapeutic drug monitoring (TDM) [14]. LZD and DAP are effective for

Gram-positive bacteria such as *Enterococcus faecium*, *Staphylococcus aureus*, *Streptococcus agalactiae*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, and others, which are resistant to other antibiotics [15]. However, LZD may induce pancytopenia, which makes it difficult to continue the administration of the drug. DAP is also used to treat various bacterial infections caused by Gram-positive bacteria, including methicillin-resistant *S. aureus* (MRSA) and vancomycin-resistant *Enterococci* (VRE) [16, 17]. As mentioned above, LZD and DAP are also known to interact with WF, and therefore, pharmacists monitor the fluctuation of PT-INR carefully and provide prescription support [18].

#### *2.1.3 Other agents*

The administration of cardioprotective drugs such as angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and/or betablockers is essential for patients awaiting HTx, and that of antiarrhythmic agents is an option to treat ventricular arrhythmias. Among these drugs, certain antiarrhythmic agents have the potential to cause drug–drug interactions, and pharmacists should pay careful attention to those.

Antiarrhythmic agents are categorized depending on their mechanism of cardiac action and certain types of arrhythmias, and the Vaughan Williams classification is the most widely recognized system. Vaugham Williams class Ib (mexiletine) and class III (AMD, sotalol) drugs are often administered to patients awaiting HTx. TDM is necessary for the optimal administration of these antiarrhythmic agents [19, 20]. Because AMD can interact with WF, pharmacists need to check side effects on the thyroid and lungs. Sotalol dosing is based on renal function, and a careful approach is recommended for initial dosing and up-titration.

#### **2.2 Patient education**

During hospitalization, patients and their families need to be educated that some foods and drinks in daily life may increase drug effects as mentioned above [7–9]. Especially, VK is a typical factor influencing the control of PT-INR in patients taking WF and is usually obtained from green vegetables and vegetable oils as well as VK supplements. In Japan, people also traditionally eat a large amount of boiled vegetables, and often eat *natto*, a traditional fermented food in Japan that produces VK in the intestinal flora. Japanese people tend to consume more VK-containing foods, and the therapeutic effectiveness of WF may be diminished by high VK intake. Therefore, HTRs are free to eat vegetables, but all HTRs are prohibited from eating *natto*, and *chlorella* and *green juice* are also sometimes prohibited throughout the dosing schedule of WF. Pharmacists explain the importance of dietary management to obtain stable PT-INR to the patients receiving VAD and their families/relatives during hospitalization [12]. Meanwhile, medical staffs cannot frequently check PT-INR and laboratory test values after discharge. To allow the patients to be aware of fluctuations in PT-INR values, pharmacists also need to educate them to monitor PT-INR by themselves using CoaguChek® and determine the WF dosage based on the results of the PT-INR scale.

A certain amount of variability can be seen among individual CoaguChek® devices, and therefore, we monitor PT-INR values calculated by blood sampling during patients' hospitalizations. Considering the difference between the CoaguChek® and PT-INR values, we are trying to obtain an optimal scale for WF adjustment.

HTx surgery is not scheduled, and there are restrictions on visiting rooms after the surgery for clean room management. Therefore, pharmacists must explain about *Role of the Transplant Pharmacist DOI: http://dx.doi.org/10.5772/intechopen.102372*

post-transplant medication from the early stage during the HTx waiting period. In addition, pharmacists aim to remove patients' anxiety about pharmacotherapy associated with the change from anticoagulant therapy under heart failure to immunosuppressive therapy and also need to facilitate the introduction of self-managed immunosuppressive medication after transplantation.

#### **3. Perioperative and postoperative HTx**

#### **3.1 Pharmaceutical management**

#### *3.1.1 Protocol preparation*

The protocol for administration of antimicrobials and immunosuppressants during the perioperative period is prepared from the day before to the day of HTx. To create it, pharmacists check the recipient's conditions such as histories of side effects and allergy, laboratory values, preoperative bacterial infection status, viral antibody titer, and current prescriptions and then discuss the need for new or continuous prescriptions. In addition, pharmacists participate in medical staff meetings to confirm donor information including viral antibody titer and preoperative cardiac function and also confirm the timing when the recipient enters into an operating room and determine whether basiliximab should be administered. Basiliximab is not approved for the treatment of HTx in Japan, but a few reports have described the use of basiliximab as beneficial after HTx [21, 22]. Therefore, we usually prepare protocols in case we might use basiliximab for HTRs [23]. The protocol prepared for antibacterial and immunosuppressive therapies created by pharmacists based on this information is shared with cardiac surgeons, cardiologists, and RTCs after approval by the physician in charge.

#### *3.1.2 Immunosuppressive agents*

Perioperative immunosuppressive therapy basically consists of a combination of three drugs: calcineurin inhibitors (CNIs), mycophenolate mofetil (MMF), and steroids. Alternative immunosuppression strategies are needed for patients with renal impairment, fatal acute cellular rejection (AMR), antibody-mediated rejection (AMR), and infections. Herein, the immunosuppressive strategies are described by dividing it into three therapies: induction, maintenance, and response to rejection.

#### *3.1.2.1 Induction therapy*

Basiliximab is a chimeric mouse-human monoclonal antibody that binds to the receptor of interleukin 2 (IL-2), inhibiting the proliferation of T cells, and is approved for the suppression of acute rejection response after renal transplantation in Japan, but not for heart transplantation. Basiliximab has been widely used as an induction therapy in renal transplantation, although the incidence of adverse events, such as cytomegalovirus (CMV) infection, malignancies, or post-transplant lymphoproliferative disorders, is of concern [24].

At NCVC, the standard immunosuppression protocol for HTRs is the regular release tacrolimus (TAC)-based triple immunosuppression therapy. Usually, TAC and MMF are introduced for HTRs immediately after postoperative decannulation and passing the swallowing test. However, TAC has side effects such as nephrotoxicity

and can exacerbate pre-HTx renal dysfunction of the HTR by increasing renal vasoconstriction caused by TAC. In such a patient, the introduction of induction therapy using basiliximab as well as two-week delayed start of administration of TAC is to be considered. To date, in our institute, the effect of induction therapy using basiliximab with delayed TAC administration on the clinical prognosis of HTRs has been verified as compared with that of a standard TAC-based triple immunosuppression therapy [23]. The former therapy might be feasible and safe for HTRs fulfilling certain inclusion criteria including renal function, sensitization for anti-human leukocyte antigen (HLA) antibody, and HTR- and donor-related risk factors, although a comprehensive evaluation of the clinical necessity of basiliximab-based induction therapy is necessary (see [23] for more detailed inclusion criteria). Basiliximab-based induction therapy is also applied to pediatric HTRs and the patients experiencing long-time aortic blockage.

#### *3.1.2.2 Maintenance therapy*

#### *3.1.2.2.1 Calcineurin inhibitors (CNIs)*

CNIs such as TAC and cyclosporine (CYA) exert their immunosuppressive effects by reducing interleukin-2 (IL-2) production and IL-2 receptor expression, leading to a reduction in T-cell activation. Briefly, TAC and CYA inhibit T-lymphocyte activation by binding to a member of the immunophilin family, FKBP12 and cyclosphilin, respectively. The complex formed by the drug-binding protein, calcium, calmodulin, and calcineurin inhibits calcineurin-mediated dephosphorylation and subsequent translocation of the nuclear factor (NF) of an activated T cell (NFAT) to the nucleus. NFAT initiates transcription of pro-inflammatory cytokines, including IL-2 and of its receptor. These CNIs also inhibit the activation of other transcription factors involved in IL-2 gene expression in T cells such as NF-kB. Thus, CNIs inhibit a variety of immune functions and have a narrow therapeutic index, meaning that lower exposure to a CNI induces organ rejection, whereas higher exposure induces serious infections and malignancies caused by overimmunosuppression. Therefore, pharmacists need to conduct TDM to adequately design dosage regimens of CNIs.

TAC and CYA are metabolized by the CYP3A subfamily, and many drug interactions with these CNIs reported in the solid organ transplant population are associated with intestinal and hepatic CYP3A. CYP3A is a most important drug metabolizing enzyme that has a wide substrate specificity, and a very large number of drugs are the substrates for this enzyme. Pharmacists routinely check newly prescribed medications in combination with CNIs, especially those on the list (**Table 1**), which are often used in HTRs at NCVC.

AMD is metabolized through the CYP3A metabolic pathway, and it has been reported that patients receiving AMD prior to transplant require a reduction of the TAC dose [25]. It is therefore necessary to check blood levels of both AMD and TAC carefully. Amlodipine, a substrate of CYP3A, is often used to control blood pressure during the perioperative period, and a careful control of the blood concentrations of TAC after the initiation of TAC is needed [26, 27]. Clotrimazole inhibits CYP3A function [28]. To date, oral clotrimazole lozenges have been used for prevention of opportunistic infections at NCVC, but we have experienced a need for dose adjustment of TAC by hospitalization when this drug is discontinued 6 months after HTx [29–31]. Herein, we have switched to oral amphotericin B for treatment, and since then, it has succeeded in maintaining stable pharmacokinetics of TAC [32]. HTRs with nontuberculous mycobacterial (NTM) disease take rifampicin (REP) and macrolides. REP induces the expression of various CYP subfamilies, whereas erythromycin and


#### **Table 1.**

*A list of medications that pay particular attention to their interaction with TAC during waiting period for HTx, at HTx surgery and post-HTx at NCVC. TAC, tacrolimus; HTx, heart transplantation; NCVC, National Cerebral and Cardiovascular Center, Japan.*

clarithromycin (CAM) among macrolides have a potential to inhibit CYP3A4 function and are metabolized by CYP3A4. The concomitant administration of these drugs can have a significant effect on the pharmacokinetics of TAC. For HTRs taking REP or CAM prior to HTx, pharmacists ask the specialists in advance to change from REP to rifabutin and from CAM to azithromycin to prevent worsening of NTM owing to immunosuppressive therapy and also consider the effect of concomitant drugs on the blood concentrations of TAC [33]. Even if the drugs are not used in combination with TAC, it should be noted that in patients taking drugs with a long half-life, such as AMD, before HTx, the drug may remain in the body for a long time post-HTx, thereby possibly affecting the pharmacokinetics of TAC.

Meanwhile, CYP3A5, as well as CYP3A4, is involved in TAC metabolism [34], and single nucleotide polymorphism in the *CYP3A5* gene, *CYP3A5\*3* (6986A>G), is associated with alteration in its metabolic activity, thereby affecting the blood concentration of TAC [35]. The *CYP3A5* genotype is a factor to be considered for TAC dose adjustment. At NCVC, pharmacists determine the *CYP3A5* genotype with the consent of the recipient [35, 36]. As shown in the **Figure 1**, compared with the frequencies found by previous studies in the Japanese population, we have not found any significant difference in the frequencies of the genotypes between *CYP3A5\*1/\*1* or *\*1/\*3* (CYP3A5 expresser) and *CYP3A5\*3/\*3* (CYP3A5 non-expresser) [37–39].

At NCVC, in the standard triple immunosuppressive therapy, TAC is generally initiated at a dose of 1 mg/day on the first or second postoperative day.

Thereafter, its dosage is adjusted to achieve an initial blood concentration range of 9–12 ng/mL within a week. Standard target trough levels of TAC to be maintained during the first year post-HTx are 9–12 ng/mL. Depending on the type of concomitant drug, the HTR's renal function, and the status of side effects, the dose of TAC is basically increased or decreased in one step with 0.2 or 0.5 mg as single dose, and in some case in two steps.

Meanwhile, as mentioned above, the total clearance of TAC in HTRs with *CYP3A\*1/\*1* or *CYP3A\*1/\*3* is considered to be higher than in those with *CYP3A\*3/\*3*. Therefore, in the former HTRs, it may be better to standardize two-step dose adjustment and also to set the starting dose to twice the standard levels, although this treatment strategy should be verified [26, 35].

#### *3.1.2.2.2 MMF*

MMF is an orally administered prodrug of mycophenolic acid (MPA), which blocks *de novo* biosynthesis of purine nucleotides and lymphocyte proliferation by

**Figure 1.**

*Distribution of genetic polymorphisms of CYP3A5 in HTRs at NCVC.HTRs, heart transplant recipients; NCVC, National Cerebral and Cardiovascular Center, Japan.*

suppressing the enzyme inosine monophosphate dehydrogenase [40]. Concerning MMF, the package insert clearly states that the dosage of MMF varies widely from 500 to 1500 mg per dose. Because the tolerated and effective doses vary from patient to patient, careful adjustment is necessary to achieve optimal therapeutic effects of MPA. In addition, patients with severe renal dysfunction need to be carefully taken care of because the blood levels of MPA can be high [41]. Immediately after HTx, the effects of heart failure are still present, and circulatory conditions are unstable. During this period, the appearance of side effects such as leukopenia should be noted. When the circulatory state has stabilized, pharmacists confirm pharmacokinetics and pharmacodynamics of MPA by area under the blood concentration-time curve (AUC) and make sure there is no rejection based on the myocardial biopsy results.

Diarrhea is one of the adverse effects observed during treatment with MMF [42, 43]. To alleviate it, Chinese herbal medicine *Hangeshashinto* is often used during cancer chemotherapy [44, 45], and it is also expected to be effective against diarrhea caused by treatment with MMF [46]. At NCVC, we additionally administer *Hangeshashinto* to patients treated with MMF, who are free from any suspected infection in the perioperative period or to post-HTx outpatients.

#### *3.1.2.2.3 Steroids*

Steroids have anti-inflammatory, immunosuppressive, and lympholytic effects by preventing the production of cytokines and vasoactive substances, including IL-1, IL-2, IL-6, tumor necrosis factor-α, chemokines, prostaglandins, major histocompatibility class II, and proteases. In HTx, methylprednisolone and prednisone are used frequently as part of the immunosuppressive regimen to prevent rejection. Intravenous methylprednisolone is administered at the initiation of the transplant procedure, and the dose is repeated until 3 weeks after the HTx. Thereafter, the steroid dose is gradually reduced until completion of 5 weeks post-HT, although methylprednisolone is switched to oral prednisolone if cardiac allograft rejection is not found at the myocardial biopsy after 3 weeks post-HTx.

Meanwhile, side effects are often a problem when using steroids, and pharmacists need to be aware of patient complaints and clinical findings. Delayed wound healing, diabetes, and gastric ulcer are often found in the postoperative acute phase, and osteoporosis, cataracts, hypertension, depression, and growth retardation are long-term problems.

At NCVC, HTRs are hospitalized for routine endomyocardial biopsies to assess graft rejection, coronary angiography, and coronary intravascular ultrasound with the development of cardiac allograft vasculopathy. If cardiac allograft rejection is not observed in the myocardial biopsy, the dose of steroid is tapered over 6–12 months before its discontinuation, except for HTRs with cardiac sarcoidosis who are treated with low-dose prednisolone. Meanwhile, if cardiac allograft rejection was detected in a regular myocardial biopsy after HTx, the patients are treated with augmented immunosuppression and intravenous steroids, and we consider the oral dose of steroid after the steroid pulse therapy.

We manage pediatric patients to decrease the dose of steroid or terminate steroid use as early as possible considering their healthy growth.

#### *3.1.2.2.4 Everolimus (EVL)*

Everolimus (EVL) is an inhibitor of the mammalian target of rapamycin (mTOR), a phosphatidylinositol 3-kinase-related kinase, and plays a central role in the regulation of many cellular functions including growth, proliferation, and survival [47]. EVL are usually introduced in HTRs during the maintenance phase after HTx and are often used by switching from or adding to MMF. Meanwhile, EVL can cause poor wound healing, and its initiation should be delayed up to about 2 months after surgery. The major reasons for the switching or adding are as follows: (1) post-transplant cardiac allograft vasculopathy (CAV) progression; (2) reduced renal function; and (3) malignant tumor complications, especially post-transplant lymphoma (PTLD). In an international consensus report, a target trough EVL concentration of 3–8 ng/mL was proposed [48], while paying attention to adverse events including hyperlipidemia, wound infection, acne-like skin lesions, and leukopenia. Meanwhile, EVL is a substrate of the CYP3A metabolic enzyme, and when used in combination with a CNI, the blood concentrations of the CNI need to be adjusted to 2/3 to 3/4 and sometimes 1/2, before concomitant use. Pharmacists herein prepare to renew the dosing regimens of EVL as well as CNI, which can contribute to reduce the burden on the physician. In the event of EVL introduction, the EVL protocol is prepared upon physician's request, and EVL blood concentration is monitored once or twice a week after EVL initiation, followed by myocardial biopsy approximately one month after EVL introduction. When preparing the EVL protocol, information on the rationale for the change of regimen

and optimal blood concentration of EVL is collected from the physician, and then the change of regimen and the schedule of visiting the hospital for the collection of blood are explained to the patient. After the start of EVL administration, there are concerns about the occurrence of stomatitis. The pharmacists instruct the HTRs to maintain the mouth clean and to use dexamethasone ointment and azulene sulfonic acid as a treatment when stomatitis occurs. The blood concentration level of TAC and EVL is carefully monitored considering the competitive interaction with FK-binding protein.

#### *3.1.2.3 Response to rejection*

Rejection after HTx includes acute rejection immediately after surgery, acute cellular rejection (ACR) that may occur within a few weeks to 2 years post-HTx, and CAV after those. It is also classified into cellular rejection, antibody-related rejection (AMR), and mixed type according to the mechanism of onset. Among them, AMR has been paid attention to during maintenance immunosuppressive therapy after HTx.

For AMR, the following treatments can be considered: (1) plasmapheresis to remove antibodies from the circulation; (2) intravenous immunoglobulin therapy and anti-CD20 monoclonal antibody (rituximab) to suppress antibody production; (3) use of corticosteroids to suppress the inflammatory response; (4) change of immunosuppressive therapy (use of cyclophosphamide and change from CYA to TAC) and/or dose adjustment of immunosuppressive agents; and (5) use of antithymoglobulin to suppress helper T cells.

#### *3.1.3 Prevention of infectious diseases*

Because HTRs receive immunosuppressive therapy, sufficient prophylactic treatment against infections is necessary soon after HTx. Perioperative antibiotic therapy is selected based on microbiologic sensitivities. Prophylactic treatment for bacterial infections includes broad-spectrum drugs against Gram-positive and Gram-negative bacterium, such as LZD and doripenem. MRSA and fungal infections may also be problematic in patients bridged from VAD to HTx. Intravenous antifungals such as micafungin (MCFG) are administered for fungal infections. MCFG intravenous infusion is changed to AMPH B gargle after passing the drinking water test and continued for 6 months after HTx. To prevent opportunistic infection, HTRs receive anti-*Pneumocystis* prophylaxis with sulfamethoxazole/trimethoprim for life, and the dose is adjusted depending on the HTR's renal function. Cytomegalovirus (CMV)-seropositive HTRs are routinely administered CMV immunoglobulin immediately after the HTx, which is continued until 5 days post-HTx. In CMV-seronegative HTRs transplanted with organs from CMV-seropositive donors (CMV mismatch), anti-CMV drugs such as ganciclovir or its prodrug valganciclovir are administered prophylactically at half the therapeutic dosage within 10 days of HTx and are continued until 1 year after the HTx at NCVC. If the results of CMV antigenemia and real-time polymerase chain reaction tests are positive, an anti-CMV drug is initiated at a therapeutic dose (900 mg/day) in cases with clinical symptoms or as preemptive therapy in asymptomatic cases when CMV DNA exceeded the threshold set for active CMV infection. The dose of the anti-CMV drug is adjusted according to the patients' conditions such as renal function.

#### **3.2 Patient education**

After HTx, immunosuppressive therapy and prevention of opportunistic infections are in essence supported by the recipient's adherence to medication, and patient

#### *Role of the Transplant Pharmacist DOI: http://dx.doi.org/10.5772/intechopen.102372*

education is essential to ensure adherence and understanding of the need for lifelong pharmacotherapy. Patient education should start during the transplant waiting period and continue after HTx until the patient can self-manage by the time of discharge.

MMF is teratogenic and requires a contraceptive period of 6 weeks after discontinuation as well as during administration. Therefore, pharmacists need to educate potentially pregnant recipients on contraception.

If HTRs wish to give birth, the use of MMF and mizoribine is avoided, and it is switched to immunosuppressive therapy based on CNI and azathioprine use because MMF and mizoribine are known to be teratogenic. In addition, ACEIs and ARBs, which are administered as antihypertensive agents and cardiac protective agents, have also been reported to cause oligoamnios or increase the risk of teratogenicity. In such a case, we consider administering methyl-dopa as an antihypertensive agent as needed and changing to nifedipine after 20 weeks of gestation [49].

#### **4. After discharge from HTx**

#### **4.1 Pharmaceutical management**

HTRs who have passed the acute postoperative stage are treated in cooperation with hospitals near their homes, taking into consideration their return to society. In our institute, pharmacists provide continuous support for the dose adjustment of immunosuppressive drugs from our hospital to the collaborating hospital after discharge through fax and e-mail communication. However, there are sometimes inter-institutional differences in the results of immunosuppressant blood concentration owing to different measurement methods. Therefore, pharmacists need to confirm the measurement method with each collaborating hospital in advance to adjust the dose of immunosuppressant accordingly.

When patients are prescribed a new drug at a hospital or clinic, they are instructed to contact the RTC and ask for instructions on whether or not they can take the prescription medications. The RTC informs the physician and pharmacist about the new prescription drugs and the patient's condition, and then the pharmacist evaluates possible interactions between the new prescription drugs with the drug the patient is taking, especially immunosuppressive drugs. If interactions that affect the efficacy of the immunosuppressive drugs are expected, the pharmacist provides and shares the information with the physician. If the physician decides that the patient needs to continue taking the immunosuppressive drugs despite the interaction, the pharmacist recommends when to check their blood concentrations as needed.

#### **4.2 Patient education**

#### *4.2.1 Patient education for therapy adherence*

The main role of transplant pharmacists after discharge of the patient from the hospital is as follows: management of immunosuppressive therapy and infections during outpatient visits; guidance to improve medication adherence; and the development of protocols for dose adjustment and change of immunosuppressive agents as renal function deteriorates and CAV occurs. In particular, when changing the immunosuppressive agents, pharmacists have to explain the need to change the drug and the accompanying need for blood sampling to the patients and also instruct the patients to contact their local pharmacies to share the new protocols.

When patients return to society, they often have difficulties in taking their immunosuppressant medications on time owing the time and means of commuting to school or work. In such a case, pharmacists support the patients by shifting the time of taking the medication and also instruct them to pay attention to the time of blood collection before taking the medicine during outpatient visits.

CYA-associated side effects include hirsutism and gingival thickening. Hirsutism has cosmetic problems, especially for women and adolescents, and may reduce adherence to medication. In addition, gingival thickening, especially in infants, interferes with the subsequent development of teeth, and in some cases, repeated gingivectomy may be required. In this case, switching from CYA to TAC is a treatment option.

#### *4.2.2 Diet and lifestyle*

Diet and lifestyle are important from the perspective of CAV prevention, and nutritionists provide the patients with guidance about these points during hospitalization. In addition, hyperglycemia and hyperlipidemia have been reported as side effects of immunosuppressive drugs, and pharmacists provide guidance on diet and lifestyle precautions from the perspective of these side effects. To avoid and reduce interaction with immunosuppressive agents, pharmacists should explain what foods HTRs need to be aware of and why and instruct them to avoid their intake. Such foods and diet are dietary supplements, herbal medicines, herbal teas, and grapefruit juice [50]. Meanwhile, immunosuppressive agents are taken as time-release drugs and should be taken continuously at a set time. For this reason, pharmacists also make HTRs understand the importance to maintain a regular life rhythm.

#### **5. Other relevant aspects**

#### **5.1 Individualized therapy**

Although various factors such as concomitant medications, diet, and lifestyle can influence the pharmacokinetics of immunosuppressive agents and WF, genetic polymorphisms also need to be taken into consideration as variable factors affecting the pharmacokinetics of immunosuppressive drugs and WF during HTx waiting time and after HTx. Pharmacists need to collect and organize information about such variable factors that cause inter-individual or intra-individual fluctuations of these drugs in HTx patients and provide them to physicians. This contributes to not only individualized therapy, but also to reduce the burden on physicians and enable task sharing.

#### **5.2 Certification of transplant pharmacists**

In the United States, the Doctor of Pharmacy (Pharm.D.) degree was established in the 1950s, and the American Society of Hospital Pharmacists introduced a residency program in the 1960s that transformed the role of pharmacists in team medicine [51]. In organ transplantation, the specialty pharmacist system was accredited in 2018, and guidelines for pharmacist services and education have been developed [52].

The Canadian Hospital Pharmacists Association reported on the expertise of transplant pharmacists in 2018 [53].

#### **6. Conclusions**

Transplant pharmacists at each hospital have built up their own expertise and are participating in medical teams at each facility, playing a role in organ transplantation. Because transplantation medicine requires individualized medical care, there are many situations in which pharmacists can contribute. As a member of the medical team, transplant pharmacists are involved in anticoagulation and immunosuppressive therapy and provide prescription support, which not only reduces the burden on physicians, but also contributes to the promotion of effective and safe use of drugs. Transplant pharmacists as well as members of NST, infection control teams, or palliative care teams can contribute to healthcare economy and healthcare safety by taking the initiative of appropriate use of agents.

Meanwhile, it is hoped that an academic society-led transplant pharmacist will be established, and that specialized transplant pharmacists can provide individualized pharmacotherapy for antibiotics, anticoagulants, and immunosuppressive agents, which have a narrow range of treatment in the field of VAD and HTx treatment in Japan as well as other developed countries.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Megumi Ikura1 \*, Kazuki Nakagita1 , Takaya Uno1 , Hiromi Takenaka1 , Sachi Matsuda1 , Miho Yoshii1 , Rikako Nagata1 , Ichiro Nakakura1 , Naoki Hayakawa1 , Tsutomu Nakamura2 , Kyoichi Wada2 and Osamu Seguchi3

1 Department of Pharmacy, National Cerebral and Cardiovascular Center, Suita, Japan

2 Education and Research Center for Clinical Pharmacy, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki, Japan

3 Department of Transplant Medicine, National Cerebral and Cardiovascular Center, Suita, Japan

\*Address all correspondence to: ikura.megumi48@ncvc.go.jp

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 11**

## Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous Population of Heart Transplant Recipients: A Pilot Study

*Francesco Lo Re, Sandro Sponga, Jacopo Angelini, Chiara Nalli, Antonella Zucchetto, Ugolino Livi and Massimo Baraldo*

#### **Abstract**

Mycophenolate mofetil (MMF) represents a cornerstone in heart transplant (HTx) treatment. The area under the 12-hour concentration-time curve (AUC0-12h) of mycophenolic acid (MPA) -MMF's active drug- is associated with treatment outcome. Nonetheless, therapeutic drug monitoring (TDM) of MPA AUC0-12h is impractical to assess in clinical practice and Limited Sampling Strategies (LSSs) represent a consolidated tool to estimate AUC0-12h. Two LSSs were previously generated in a selected cohort of HTx recipients treated with MMF and cyclosporine (CsA). This pilot study aimed to test these LSSs in a cohort of non-selected HTx recipients treated with MMF combined with CsA or tacrolimus (TAC). Complete PK profile was performed in 40 adults HTx recipients. MPA-AUC0-12h was estimated by two algorithms, LSS3 and LSS4, based on 3 and 4 time-points. The evaluation was made through linear regression and Bland-Altman analyses. Both LSS3 and LSS4 tended to underestimate the value of MPA-AUC0-12h (mean percentage prediction error, MPE%: 6.0%; and 4.8%, respectively). Nonetheless, high correlations (*r*: 0.92 and 0.94, respectively) and goodness of fit of linear regression models (R2 : 0.84 and 0.88, respectively) emerged for both LSSs. A study with a wider and more homogenous sample size should be performed to support these results.

**Keywords:** heart transplantation, immunosuppressive treatment, therapeutic drug monitoring, treatment efficacy, rejection prevention

#### **1. Introduction**

Mycophenolate Mofetil MMF (CellCept; Roche, Basel, Switzerland) is a widely prescribed drug as part of maintenance immunosuppressive regimen after heart transplant (HTx) [1]. It is frequently administered in association with calcineurin inhibitors (CNIs) like cyclosporine (CsA), tacrolimus (TAC), and prednisone.

MMF is a pro-drug that, after oral administration, is rapidly hydrolyzed to its active form, mycophenolic acid (MPA), by esterases mainly in the gastrointestinal wall, blood, and liver, but also in other tissues [2]. MPA is a selective, potent and reversible inhibitor of inosine-50 -monophosphate dehydrogenase (IMPDH), a key enzyme of the *de novo* purine synthesis. This block causes the arrest of the proliferation of T- and B-cells [2]. In addition to this major immunosuppressive mechanism, MPA could cause the alteration of lymphocyte and monocyte recruitment, adhesion, and penetration. Furthermore, exposure to MPA could result in the apoptosis of activated human T lymphocytes, and the reduction of cytokine production. Moreover, it has been evidenced an antiproliferative effect on monocytes, fibroblasts, endothelial cells, mesangial cells, and smooth muscle cells. Nonetheless, MPA could inhibit mesangial matrix expansion, and alter the cytoskeletal organization [3, 4]. Some of these effects, including the reduction of important lymphocyte cell surface antigens expression, are independent from IMPDH inhibition [5, 6].

Generally, MMF is prescribed at a fixed dose, but there are several pharmacokinetic (PK) factors that could affect its efficacy. After MMF administration, MPA shows non-linear absorption kinetics, and a complex inter-patient and intra-patients PK variability [7], that could be attributable to MPA enterohepatic circulation (EHC), graft function, genetic factors, changes in plasma protein binding, and drug–drug interactions. MPA time to reach the plasma maximum concentration (Tmax) occurs after 1–2 hours after dosage [8].

MPA presents a higher bioavailability, ranging from 80.7–94% [8]. In blood, MPA widely binds serum albumin, from 97–99% in patients with normal renal and hepatic function. Consistently, it has been evidenced that hypoalbuminemia could increase MPA free fraction *in vitro* [9] and *in vivo* [10]. In particular, an increase of 2.2-fold of MPA free fraction emerged *in vitro* when MPA albumin was reduced from 41.4 g/L to 20.7 g/L, and a further increase of 41-fold when albumin was reduced to 0.07 g/L [9]. In a study including 42 adult kidney transplant recipients, a relationship between low serum albumin and an increased MPA free fraction was reported [10]. The authors identified a threshold of 31 g/L below of which MPA free fraction was considered to be significantly elevated, suggesting that the Therapeutic Drug Monitoring (TDM) of MPA free fraction could be recommended in patients with this clinical condition [10].

MPA is mainly metabolized in liver, kidney, and gastrointestinal tract by uridine 50 -diphospho-glucuronosyltransferases (UGTs). The major metabolite of MPA, 7-O-MPA-glucuronide (MPAG), is inactive but it is present in the plasma at higher concentrations than MPA. MPAG is excreted into the urine via active tubular secretion and into the bile by multi-drug resistance protein 2 (MRP-2), and at the gastrointestinal level MPAG could be de-conjugated back to MPA by gastrointestinal flora and then reabsorbed in the colon, resulting in a secondary plasma peak between 6 and 12 hours after oral administration. This may contribute to the 30–40% of MPA exposure. Severe renal impairment, liver disease, and hypoalbuminemia could affect MPA exposure [11]. The co-administration of CsA, by inhibiting the MRP-2 mainly in the gastrointestinal tract, causes a reduction of MPA EHC, resulting in an approximately 30–40% lower MPA exposure than when MMF is administered in combination with TAC [2, 8, 12]. Furthermore, it has been evidenced that CsA administration could affect MPA Clearance (Cl) [13]. Moreover, corticosteroids may reduce the exposure of MPA by inducing the expression of UGTs [8].

For these reasons, the execution of TDM could be an effective strategy to maximize the efficacy of the treatment also reduce the risk of toxicity. Several studies have suggested the importance of MPA TDM in renal and heart transplants recipients [14–16]. The best PK parameter correlating with the efficacy of treatment is represented by MPA's area under the plasma concentration-time curve from 0 to 12 hours (MPA AUC0-12h) [11, 17] and several studies show that MPA plasma levels correlate to risk of rejection [18, 19]. The therapeutic range has been well determined in renal transplant recipients (30–60 mg h/L) [20], and some authors suggested similar therapeutic thresholds on MPA-AUC0-12h also in HTx [21, 22].

The entire MPA AUC0-12h is difficult to calculate in clinical practice, due to its costly and laborious assessment. On the contrary, the single time-point measurement is the easiest for sampling, but it is not sufficiently predictive of patient outcome [20], taking also in consideration that MPA is characterized by >10-fold range variation in MPA AUC0-12h dose-normalized among patients undergoing heart or renal transplantation [23, 24].

Limited Sampling Strategies (LSSs) represent the most relevant assessment in solid organ transplantation for dosage individualization, that could overcome this problem [20]. LSSs are algorithm-based strategies able to predict the entire AUC0-12h without the necessity of sampling all the time-point concentrations after drug administration, but limiting the sampling to a reduced number of measurements, usually three timepoints or even fewer. They can be developed by two main methods represented by multiple linear regression (MLR) or by with maximum a posteriori Bayesian estimation (MAP-BE).

MLR represents the simplest technique to develop an LSS. It requires statistical knowledge and the main strength of this approach is the adhesion to the sampling time.

On the other hand, developing an LSSs by maximum a posteriori Bayesian estimation (MAP-BE) is more complex because specialized PK modeling software knowledge is required.

From a methodological point of view, LSS should be generated on a cohort of patients (*training group*) and then validated in the second cohort of patients (*validation group*) to be used in clinical practice [25]. In the case of MLR LSSs, the relationship between the observed AUC0–12h and the estimated blood concentration-time points must be determined in the *training group* through linear regression, considering AUC0–12h as the dependent variable and the blood concentrations at each time point as the independent variables.

To exclude biased results, the LSS performance should be assessed in the *validation group* evaluating the mean prediction error or bias and the root mean squared prediction error or precision, as well as the median prediction error and the median absolute prediction error [26]. These same figures can be also calculated based on percentage prediction error, and expressed in percentages, to be more easily interpretable in the clinical contest as suggested by Baraldo et al. [25]. In both cases, the values of these parameters are inversely and proportionally linked to the LSS prediction. In the end, the correlation coefficient (*r*) and the coefficient of determination (R2 ) between the estimated and the observed AUC0-12h must be assessed.

Recently, Baraldo et al. reviewed the state of the art of MPA LSSs in HTx recipients [25]. In the last few years, the immunosuppression therapy after HTx has changed, with the massive use of TAC compared to CsA, in combination with MMF and corticosteroids.

This pilot study aimed to test, in a heterogeneous cohort of patients treated with MMF and CSA or TAC, two algorithms of LSSs previously generated by Baraldo et al. [27, 28] in a selected cohort of HTx recipients treated with MMF and CSA. These algorithms were selected due to their good performance [28] and given the hypothesis that the LSSs sampling time point schedule was able to determinate MPA AUC0-12h even when MMF was administrated combined with TAC.

If this pilot study reports positive results, the generation of new LSS in a population of HTx treated with MMF and TAC would not be required.

#### **2. Methods**

#### **2.1 Study characteristics**

This is a pilot observational, retrospective, cohort study. The study was performed at the University Hospital of Udine, in Italy. The study was approved by the Internal Review Board (I.R.B.) of the Commission for the Experimentation and Protection of Human Subjects of the Department of Medical Area of the University of Udine with the protocol number: 036/2020\_IRB.

The study included 40 HTx recipients previously treated as per standard clinical practice with MMF and CsA or MMF and TAC, and prednisone, at the University Hospital of Udine, and routinely monitored for MPA quantification in the period starting from the 01st/01/2011 up to the 31st/12/2019. The patients included in the study were HTx recipients, aged 18 years old or more, and treated with MMF and either CsA or TAC and prednisone. Patients treated with immunosuppression drugs other than MMF, CsA and TAC, or with the absence of necessary information for the study in the clinical records or with the absence of informed consent for clinical, epidemiological research, training and study of pathologies were excluded from the study. All consecutive HTx recipients in the study period who met inclusion/exclusion criteria were included in the analysis.

All HTx recipients received a standard triple immunosuppressive therapy: MMF in combination with CsA or TAC and prednisone. The posology regimen of MMF varied from 1000 to 3500 mg/day, with a mean of 1785.5 mg/day ( 553.4). While the mean CsA dose was 3.0 mg/kg/day ( 1.3) p.o. in 2 divided doses, mean TAC dose was 0.1 mg/kg/day (0.06). Patients treated with prokinetic drugs, resins or other drugs known to interfere with MPA PK, other than prednisone, were excluded from the analysis.

#### **2.2 PK profiles of mycophenolate mofetil**

A complete PK profile was available for the 40 HTx recipients included in the present analysis. Patients had been asked to take their usual morning dose of MMF after having a standard meal. Patients had not changed the therapeutic regimen for 30 days and had been at a steady state for MMF. Eight venous samples had been collected for the analysis of MPA plasma concentrations. For MPA assays, blood samples had been collected in EDTA tubes at 0 (pre-dose), 0.5, 1.25, 2, 4, 6, 8, and 12 hours after the morning dose. Separation of plasma was performed immediately in a centrifuge at 4°C. Plasma MPA concentration was measured using validated High Pressure Liquid Chromatography with UV Detector (HPLC/UV) method [23], that ensure to achieve an analytical precision and accuracy that fulfill the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) recommendations [20]. The laboratory reported the following parameters for the HPLC/UV method used for MPA quantification: limit of detection, 0.1 μg/mL; linearity, 0.1–40 μg/mL (R<sup>2</sup> : 0.9988); intrabatch imprecision (CV), 3.15%, 1.55%, and 1.76% *Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous… DOI: http://dx.doi.org/10.5772/intechopen.102412*

at MPA plasma concentrations of 1.5, 5.0, 15.0 μg/mL, respectively; interbatch imprecision (CV), 3.41%, 3.21%, and 1.92% at MPA plasma concentrations of 1.5, 5.0, 15.0 μg/mL, respectively; overall inaccuracy (% Bias) of the procedure, ranged from 8.7% to 13.6%. MPA AUC0–12h had been calculated by the linear trapezoidal rule.

#### **2.3 Algorithms evaluation**

The two algorithms used for MPA AUC0-12h evaluation were the followings:

*LSS***3** : *MPA AUC***<sup>0</sup>**�**12***<sup>h</sup>* ¼ **5***:***568** þ **0***:***902** � *C***<sup>1</sup>***:***25***<sup>h</sup>* þ **2***:***022** � *C***2***<sup>h</sup>* þ **4***:***594** � *C***6***<sup>h</sup> LSS***4** : *MPA AUC***<sup>0</sup>**�**12***<sup>h</sup>* ¼ **3***:***800** þ **1***:***015** � *C***<sup>1</sup>***:***25***<sup>h</sup>* þ **1***:***819** � *C***2***<sup>h</sup>* þ **1***:***566** � *C***4***<sup>h</sup>* þ **3***:***479** � *C***6***<sup>h</sup>*

According to Sheiner and Beal, to assess the bias of the LSSs, we calculated Mean Percentage Prediction Error (MPE%) and the Median Percentage Prediction Error (MPPE%) [26]. To assess precision was calculated Root Mean Squared Percentage Prediction Error (RMSE%) and theMedian Absolute Percentage Prediction Error (MAPE%) [26].

The MPE%, MPPE%, RMSE% and MAPE% were calculated as follows: Bias:

$$\text{MPE\%} = mean \left( \frac{predicted \, AUC\_{0-12h} - measured \, AUC\_{0-12h}}{measured \, AUC\_{0-12h}} \times 100\% \right) \tag{1}$$

$$\text{MPPE\%} = median \left( \frac{predicted \, AUC\_{0-12h} - measured \, AUC\_{0-12h}}{measured \, AUC\_{0-12h}} \times 100\% \right) \tag{2}$$

Imprecision:

$$RMSE \%= \sqrt{mean \left(\frac{predicted \ AUC\_{0-12h} - measured \ AUC\_{0-12h}}{measured \ AUC\_{0-12h}} \times 100\% \right)^{2}} \quad \text{(3)}$$

$$(\text{predicted } AUC\_{0-12h} - measured \ AUC\_{0-12h})$$

$$\text{MAPE\%} = median \left( \frac{|\text{predicted AUC}\_{0-12h} - measured \, AUC\_{0-12h}|}{measured \, AUC\_{0-12h}} \times 100\,\text{\%} \right) \tag{4}$$

For bias, we set the limit of 15%, while for imprecision the limit was set at 20%. The percentage of estimated AUC0-12h between 75–125% of the observed AUC0-12h was also calculated.

To compare our results to an already validated algorithm, we tested one other LSS equation developed in HTx by Kaczmareck et al. [29]:

### *LSSKazmareck* : *MPA AUC***<sup>0</sup>**�**12***<sup>h</sup>* ¼ **1***:***65** � *C***<sup>0</sup>***:***5***<sup>h</sup>* þ **4***:***74** � *C***2***<sup>h</sup>*

#### **2.4 Statistical consideration**

Descriptive statistical analyses were conducted for all the study variables, reporting position and variability indexes (e.g., mean and standard deviation, SD) for quantitative variables. Differences between groups were evaluated using the Fisher's exact test for nominal variables and the Student's T-test for quantitative variables, and considering as statistically significant a p-value <0.05.

The two methods of LSS were validated by using both linear regression and Bland–Altman analysis, as recommended by the literature [26, 30]. All the analyses were performed with Medcalc Software version 19.7.2 ® (Med-Calc Software, Ostend, Belgium®). Pearson's linear correlation coefficient (*r*) was calculated using linear regression (considering the following categories for the absolute value |*r*|: <0.50 weak correlation, 0.50–0.80 moderate correlation; >0.80 strong correlation). The determination coefficient (R2 ) was also reported to assess the goodness of fit of the linear models. Bland–Altman analysis was used to evaluate the agreement between the predicted AUC0–12h and the measured AUC0–12h.

#### **3. Results**

#### **3.1 Patients characteristics**

The main characteristics of study patients are reported in **Table 1**.

All patients were Caucasian and most of the analyzed patients shown normal renal and hepatic functionality. Patients treated to CsA- or TAC-based maintenance immunosuppression were comparable for most of the baseline characteristics, including age, body mass index (BMI), MMF administered dose, renal and hepatic function, except for sex, bilirubin, post transplantation time, MPA AUC0-12h and MPA C0. A number of 15 acute cell rejections occurred after a median time of 8.95 months from transplantation, especially in the patients group treated with MMF-CsA than in the MMF-TAC group (87% vs. 13%, respectively). According to the International Society for Heart and Lung Transplantation, the overall rejections were classified as follows: 8 GRADE 1R (55%), 5 GRADE 2R (33%) and 2 GRADE 3R (13%) [31]. No patients reported any episodes of diarrhea.

#### **3.2 Method results**

In the whole cohort of patients, a low tendency to underestimation of the value of MPA AUC0-12h by both LSS3 and LSS4 emerged evaluating MPE% for mean values (6.0% and 4.8%, respectively) and MPPE% for median values (3.8% and 1.1%, respectively). The precision of LSS3 and LSS4 was acceptable, by evaluating RMSE% for mean values (19.6% and 16.2%, respectively) and MAPE% for median values (13.5% and 11.0%, respectively). The percentages of MPA AUC0-12h predicted by LSS3 and LSS4 within the 25% of the MPA AUC0-12h full value was 73% and 80%, for LSS3 and LSS4, respectively.

Linear regression and Bland–Altman analyses evidenced that both LSS3 and LSS4 methods can effectively predict the values of MPA AUC0-12h. The value of *r* stated for both LSSs methods a strong correlation between the measured MPA AUC0-12h and the AUC0-12h predicted by both LSSs methods (*r*: 0.92 and 0.94 for LSS3 and LSS4, respectively). Finally, the R2 (0.84; 0.88, for LSS3 and LSS4, respectively) indicates high goodness of fit of the regression line for both methods. The results are shown in **Figure 1a** and **b**. The Bland–Altman plots (**Figure 2a** and **b**) showed that the data were arranged almost totally within the range mean +/1.96\*SD. The visual inspection of the plots does not reveal any particular pattern, thus excluding other types of bias. This was also assessed by analyzing the linear dependence of the dots in the Bland Altman plot using linear regression, reporting the following results for LSS3 and LSS4 respectively (*r* = 0.51 and 0.55; R<sup>2</sup> : 0.26 and 0.30). These results do not indicate linear dependence.

A subgroup analysis was also conducted stratifying the patients for the co- treatment.


*Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous… DOI: http://dx.doi.org/10.5772/intechopen.102412*

*a p-values of 2-sided Fisher's exact test for nominal variables or T- test for quantitative variables.*

*b Evaluated by Cockcroft-Gault adjusted for body weight.*

*c Evaluated by CKD-EPI Equation.*

*Data are reported as mean standard deviation, if not otherwise specified.*

*AUC0-12h: Area under the plasma concentration-time curve from zero to 12 h; ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; Bas: Basophils; BMI: Body Mass Index; C0: pre-dose measurement; CsA: Cyclosporine; CrCl: Creatinine Clearance; Eos: Eosinophils; GFR: Glomerular Filtration Rate; Hb: Hemoglobin level; Lymph: Lymphocytes; Mono: Monocytes; MMF: Mycophenolate Mofetil; MPA: Mycophenolic Acid; Neutro: Neutrophils; RBCs: Red Blood Cells; TAC: Tacrolimus; WBCs: White Blood Cells.*

**Table 1.**

*Patients baseline demographical and clinical data, overall and according to the type of treatment.*

#### **Figure 1.**

*Linear regression scatters plot of MPA AUC0-12h predicted versus MPA AUC0-12h measured, when MPA AUC0-12h predicted was calculated with LSS3 (A) and LSS4 (B) (n = 40 PK profile).*

#### **Figure 2.**

*Bland–Altman plots comparing MPA AUC0-12h predicted – MPA AUC0-12h measured and the average of MPA AUC0-12h predicted and MPA AUC0-12h measured, when MPA AUC0-12h predicted was calculated by LSS3 (A) and LSS4 (B) respectively (n = 40 PK profile).*

Among 28 patients treated with MMF and CsA, the bias was acceptable, evaluating MPE% for mean values (0.5% and 0.3%) and MPPE% for median values (2.3% and 0.7%) for LSS3 and LSS4, respectively. Analogously, the precision was acceptable evaluating RMSE% (18.6% and 14.8%) and MAPE% (12.4% and 9.7%), for LSS3 and LSS4, respectively. The percentages of MPA AUC0-12h estimated by LSS3 and LSS4 within the 25% of the MPA AUC0-12h full value were 79% and 86%, respectively.

Finally, in the sub-group of 12 patients treated with MMF and TAC, these same features were the followings: MPE% = 18.9% and 15.3%; MPPE% = 19.9% and 14.0%; RMSE% = 21.7% and 19.2%; MAPE% = 19.0% and 14.0%, for LLS3 and LSS4, respectively.

The percentage of MPA AUC0-12h predicted within the 25% of the measured MPA AUC0-12h full value: 58% and 67%, for LSS3 and LSS4 respectively.

Despite the very low number of patients, also the linear regression analyses executed on the two subgroups of patients evidenced good results.

In the MMF and CsA group the results were the followings: *r =* 0.83 and 0.89; R2 = 0.70 and 0.79, for LSS3 and LSS4 respectively; while in the MMF and TAC group *Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous… DOI: http://dx.doi.org/10.5772/intechopen.102412*


*AUC0-12h: Area under the plasma concentration-time curve from zero to 12 h; CsA: Cyclosporine; LSS3: Limited Sampling Strategy based on 3 concentration sampling points; LSS4: Limited Sampling Strategy based on 4 concentration sampling points; MAPE%: Median Absolute Percentage Prediction Error; MMF: Mycophenolate Mofetil; MPA: Mycophenolic Acid; MPE%: Mean Percentage Prediction Error; MPPE%: Median Percentage Prediction Error; RMSE%: Root Mean Squared Percentage Prediction Error; R2 : coefficient of determination; TAC: Tacrolimus.*

#### **Table 2.**

*Predictive performance of LSS3 and LSS4 in the estimation of the observed MPA AUC0-12h.*

these were the results: *r* = 0.93 and 0.93; R<sup>2</sup> *=* 0.87 and 0.86, for LSS3 and LSS4 respectively. All these results are summarized on **Table 2.**

The analysis of Kaczamarek LSSs applied to our patient's data reports the following results: *r* = 0.70; R2 = 0.49; MPE% = 11.4% and RMSE% = 66.1% in the overall population. By applying these LSSs in the TAC subgroup of patients, we evidenced the following results: *<sup>r</sup>* = 0.69; R2 = 0.48; MPE% = 6.2% and RMSE% = 32.1%.

#### **4. Discussion**

The importance of MPA TDM for renal transplant patients is known, but its execution on HTx patients in clinical practice is still debated [17]. Specific large prospective randomized trials should be conducted, but the considerable inter- and intra-patient variability of MPA after organ transplantation suggest MPA TDM to optimize MPA exposure.

The systematic review regarding MPA TDM in HTx reported by Zuk et al. suggests that the relationship between MPA levels and the efficacy of the treatment in terms of allograft rejection in HTx patients is not defined, but LSS may be a better assessment strategy to prevent rejection than a single-time point model [32]. An LSS can be generated using two main methods: MAP-BE method and MLR analysis.

In the first case, any recorded patient sample is compared with data derived from the population PK study, and the covariates can be continually improved by updating the PK population data. The main advantage of the first approach is represented by the flexibility in the timing of the samples as recently demonstrated by Woillard et al. [22]. The main limit of this approach is represented by the employment of complex and specific software, requiring skilled professionals.

On the contrary, multiple regression analysis is simpler, but adherence to the sampling time is mandatory to apply the algorithms in clinical practice. To our knowledge, up to now, few LSSs were developed in HTx, and most of them were generated in patients treated with MMF and CsA [25]. Only three studies focused on LSSs in HTx recipients treated with MMF and TAC [29, 33, 34].

Xiang et al. [33] generated an LSS for the estimation of MMF dispersible tablets combined with TAC in 30 Chinese HTx patients. The comparison of MPA PK among MMF dispersible tablets and MMF did not show significant differences. The LSS with the best performance was the following: MPA AUC0-12h = 8.424 + 0.781 C0.5h + 1.263 C2h + 1.660 C4h + 3.022 C6h (R2 = 0.844). The performance of this LSS can be considered comparable with our algorithms and both contain the C6h sample timing point improving the MPA AUC0-12h estimation thanks to the inclusion of the typical secondary peak of MPA, minimizing the risk of MPA AUC0-12h underestimation. Nevertheless, this LSS was developed in Chinese patients so it could not properly fit the Caucasian population, although literature does not suggest this hypothesis [35]. Moreover, these LSSs were developed analyzing the plasma timing point by Liquid Chromatography with tandem mass spectrometry (LC/MS–MS), so they cannot be easily transferred in that laboratories which employ HPLC/UV methods.

Kaczmarek et al. [29] generated different LSSs in 28 HTx recipients treated with MMF and TAC. The best LSS was obtained using 4 sampling points: MPA-AUC0-12 <sup>h</sup> = 1.25 C1h + 5.29 C4h + 2.90 C8h + 3.61 C10h (R2 = 0.95). The studied population is comparable to our population. Also, in this case, it can be seen that by sampling the timing point after several hours from MMF administration, a better MPA-AUC0-12h estimation can be achieved. These LSSs show an optimal performance, but it is based on a demanding sampling schedule that can be applied only on hospitalized patients, thus excluding the outpatient settings.

For this reason, authors proposed two different and more practical LSSs represented by: MPA AUC0-12h = 1.09 C0.5 + 1.19 C1h + 3.60 C2h (R2 = 0.84) and MPA AUC0-12h = 1.65 C0.5h + 4.74 C2h (R2 = 0.75). Due to the missing data about the C1h in our population, we test the second LSS. The performance was not acceptable for the use in clinical practice as compared to our algorithms. This could be due to the absence of the C6h sampling time point, resulting in MPA AUC0-12h underestimation.

Wada et al. [34] generated an LSS in 11 Chinese HTx recipients treated MMF and TAC approximately 9 months after transplantation. In this case, the author used the same analytical method, pharmacokinetic and statistical approaches.

They generated a 3-point model LSS based on C1h, C2h and C4h: MPA AUC0-12h = 23.56 + 1.05 C1h + 1.25 C2h + 2.53 C4h (R<sup>2</sup> = 0.73), with an MPE% of 2.73%. The results of Wada's study should be taken with caution because of the limited number of enrolled patients and the ethnic difference that could influence MPA PK.

On the other hand, Pawinski et al. proposed an accurate LSS in HTx patients treated with MMF (and CsA) [36] is based on 3 sampling time-points 2 hours after drug administration. The LSSs developed was the following: MPA AUC0-12h = 9.69 + 0.63 C0.5h + 0.61 C1h + 2.20 C2h. It showed a good performance (R2 = 0.84), and for its sampling schedule it can be applied in the outpatient setting. Nevertheless, this LSS was generated on the patient in combination therapy with MMF and CsA. For this reason, this algorithm could be acceptable in patients co-treated with CsA because of its effect on reducing the typical MPA secondary peak, affecting MPA EHC [2]. Moreover, the authors developed an algorithm including the C6h blood sample. It presented a similar R2 and can be considered more predictive of the entire AUC0-12h because it can describe the typical MPA secondary peak that occurs approximately 6 to 12 hours after MMF oral dose administration, thus affecting global MPA exposure.

In our study the two evaluated LSSs reveal to be sufficiently precise and accurate for the estimation of the entire MPA AUC0-12h **Figure 1**. The major thesis that allows the application of these LSSs in this population is the presence of C6h that offers the

*Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous… DOI: http://dx.doi.org/10.5772/intechopen.102412*

opportunity to estimate MPA PK accurately in both immunosuppressive regimens, even if it is not easy to apply in the outpatient setting.

This study has several limitations: 1) the whole study group was mainly composed by men, whereas, the small subgroup of patients treated with TAC included a high percentage of women. However, it has been demonstrated that MPA PK is not influenced by sex in solid organ recipients [8, 37], even if Tornatore et al. [38] showed differences in MPA and MPAG PK related to sex among stable renal transplant recipients receiving enteric-coated mycophenolate sodium combined with TAC.; 2) in this pilot study, the sample size of the MMF and TAC group was smaller than MMF and CsA group; 3) MMF and TAC group presented a higher C0 and MPA AUC0-12h. However, exposure to MPA when MMF is in combination therapy with CsA is approximately 30–40% lower than when given in monotherapy or with TAC [8, 39]; 4) the MMF and TAC group presented a lower level of bilirubin. Bilirubin could displace MPA from albumin binding sites, affecting MPA exposure [40]. However, this effect is limited to only patients presenting hyperbilirubinemia, and could be detected only when the free drug is measured [40]; 5) TDM was not planned to be executed at the same time for all enrolled patients but it was executed by clinical decision. This can be a source of bias, because it is known that the exposition of MPA AUC0-12h could vary extensively after HTx [11]; 6) furthermore, co-medications commonly used in clinical practice could alter MPA exposure [8, 11] . However, as shown in **Table 1**, the major clinical parameter, including age, BMI, liver and renal function between the two treatment groups were statistically comparable.

#### **5. Conclusion**

In this pilot study, two LSSs resulted to be sufficiently precise and accurate to predict MPA AUC0-12h in a heterogeneous cohort of HTx patients. This study confirmed that the two LSSs, generated in HTx recipients treated with MMF and CsA could be used also in patients treated with MMF and TAC, in particular on in hospitalized patients in the first period after HTx and in outpatients with suspected toxicity or at high risk of organ rejection with considerable social, healthcare and economic advantages.

These results suggest to confirm this hypothesis in a prospective study with a wider cohort of HTx recipients, treated mainly with MMF and TAC, and with a pre-planned TDM.

#### **Acknowledgements**

The authors thank the healthcare staff of the Clinical Pharmacology and Toxicology Institute "University Hospital Santa Maria della Misericordia", Udine, Italy, for the collection of blood samples and the analyses performed.

#### **Conflict of interest**

The authors declare no conflict of interest.

The authors received no specific funding from any organization for this work.

#### **Thanks**

The authors thank Miss. Enza Pincente for the English revision of the Manuscript.

### **Nomenclature**


*Limited Sampling Strategies to Monitoring Mycophenolic Acid Exposure in a Heterogeneous… DOI: http://dx.doi.org/10.5772/intechopen.102412*

#### **Author details**

Francesco Lo Re<sup>1</sup> , Sandro Sponga2,3, Jacopo Angelini1 , Chiara Nalli<sup>2</sup> , Antonella Zucchetto<sup>4</sup> , Ugolino Livi2,3 and Massimo Baraldo1,3\*

1 Clinical Pharmacology Institute, "Azienda Sanitaria Universitaria Friuli Centrale (ASU FC)", Udine, Italy

2 Department of Cardiothoracic Surgery, "Azienda Sanitaria Universitaria Friuli Centrale (ASU FC)", Udine, Italy

3 Department of Medical Area (DAME), Udine University, Udine, Italy

4 Scientific Directorate, "Centro di Riferimento Oncologico di Aviano (CRO), IRCCS", Aviano (PN), Italy

\*Address all correspondence to: massimo.baraldo@uniud.it

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 6
