Differences in Peripheral Blood Lymphocytes between Brand-Name and Generic Tacrolimus Used in Stable Liver Transplant Recipients

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1 Originl Pper Med Princ Prct 217;26: DOI: /5561 Received: June 22, 216 Accepted: Jnury 9, 217 Published online: Jnury 9, 217 Differences in Peripherl Blood Lymphocytes between Brnd-Nme nd Generic Tcrolimus Used in Stble Liver Trnsplnt Recipients Jong Mn Kim Choon Hyuck Dvid Kwon Je-Won Joh Dong Hyun Sinn b Gyu-Seong Choi Je Berm Prk Eun-Suk Kng c Suk-Koo Lee Deprtment of Surgery, b Division of Gstroenterology, Deprtment of Medicine, nd c Deprtment of Lbortory Medicine nd Genetics, Smsung Medicl Center, Sungkyunkwn University School of Medicine, Seoul, Republic of Kore Significnce of the Study In this study, peripherl blood lymphocytes were compred between stble liver trnsplnt recipients receiving brnd-nme nd generic tcrolimus. The level of CD+Foxp3+ T cells ws higher in the brnd-nme thn in the generic tcrolimus group fter trnsplnttion. This study found tht brndnme tcrolimus could hve more potentil immunosuppressive ctivity thn generic tcrolimus regrding the contribution of CD+Foxp3+ T cells to grft tolernce in liver trnsplnt recipients. Keywords Liver trnsplnttion γδt cells Regultory T cells Stble grft function Living donors Abstrct Objectives: In this study, peripherl blood lymphocytes were compred between brnd-nme nd generic tcrolimus group in stble liver trnsplnt recipients. Subjects nd Methods: Sixteen ptients who underwent ABO-comptible living donor liver trnsplnts between 2 nd 213 nd hd stble grft function were included in this study. Ten ptients received brnd-nme tcrolimus nd 6 ptients received generic tcrolimus. CD3, CD, CD, γδ, CD+FoxP3+, nd CD3 CD56+ T cells were nlyzed in peripherl blood obtined preopertively nd,,, nd 2 fter liver trnsplnttion. Ctegoricl vribles were compred using χ 2 test or Fisher exct test, nd continuous vribles were compred using the Mnn-Whitney U test. Results: Regrding the bseline nd periopertive chrcteristics, there were no sttisticlly significnt differences between the 2 groups. Immunosuppression lso ws not different. Subtype nlysis of T-cell popultions crried out in prllel showed similr levels of CD3, CD, CD, nd γδt cells with brnd-nme tcrolimus nd generic tcrolimus in stble liver trnsplnt recipients. However, the levels of CD+Foxp3+ nd CD3 CD56+ T cells were higher in the brnd-nme tcrolimus group thn in the generic tcrolimus group fter trnsplnttion ( p <.5). Conclusions: The level of CD+Foxp3+ T cells ws higher in the brnd-nme tcrolimus group thn in the generic tcrolimus group fter trnsplnttion. This finding showed tht brnd-nme tcrolimus could hve more potentil immunosuppressive ctivity thn generic tcrolimus regrding the contribution of CD+Foxp3+ T cells to grft tolernce in liver trnsplnt recipients. 217 S. Krger AG, Bsel E-Mil krger@krger.com S. Krger AG, Bsel This is n Open Access rticle licensed under the terms of the Cretive Commons Attribution-NonCommercil 3. Unported license (CC BY-NC) ( pplicble to the online version of the rticle only. Distribution permitted for non-commercil purposes only. Prof. Je-Won Joh, MD, PhD Deprtment of Surgery, Smsung Medicl Center Sungkyunkwn University School of Medicine Irwon-Ro 1, Gngnm-Gu, Seoul, (Republic of Kore) E-Mil smsung.com

2 Introduction Tcrolimus is one of the most commonly used medictions for the prevention of liver llogrft rejection. Tcrolimus cts s n immunomodultory gent by blocking the trnscription of the gene encoding interleukin-2 (IL- 2) tht is essentil for T-cell-medited immune response [1, 2]. Most countries hve tried to reduce the cost of cre for trnsplnt ptients, but the finncil burdens of immunosuppressive therpy remin high [3]. This sitution hs led to the development nd wide usge of generic tcrolimus, which hs met ll stndrds for bioequivlence nd is therpeuticlly equivlent to brnd-nme tcrolimus [, 5]. Mny recipients hd received generic tcrolimus fter the expirtion of the tcrolimus ptent. Generic drugs offer significnt cost svings for liver trnsplnt progrms nd recipients; however, there hs been considerble concern mong trnsplnt heptologists nd ptients bout the equivlence of the generic nd brndnme forms [3, 6]. Given the lck of dverse events reported nd the ssocited cost svings, conversion from brnd-nme to generic tcrolimus hs been encourged in orgn trnsplnttion []. αβt lymphocytes ply centrl role in severl experimentl models of llogrft rejection nd tolernce, cting s both effector nd suppressor T cells [7]. Mny studies hve demonstrted tht the T helper (Th)1/Th2 cell blnce [], nturl killer (NK) cells [9], NKT cells [1], Foxp3+ regultory T cells (Tregs) [11], nd Th17 cells [] re involved in grft function such s rejection or tolernce. Recently, γδt cells were lso reported to be ssocited with liver llogrft tolernce [13]. Humn γδt cells re composed of 2 min subsets, such s Vδ1 nd Vδ2, ccording to the rerrnged Vδ chin. Vδ1 γδt cells, which re predominnt in the spleen, intestine, nd liver, possess regultory nd effector properties, but Vδ2 γδt cells, which re the min subset of γδt cells in the peripherl blood, possess defense properties ginst pthogens [7]. Severl studies hve reported tht prepondernce of Vδ1 γδt cells is ssocited with liver llogrft cceptnce [1 16]. Some studies hve described the sfety nd efficcy of generic tcrolimus in liver trnsplnt recipients [5, 17]. However, the nlysis of peripherl lymphocytes in blood between ptients treted with brnd-nme nd generic tcrolimus hs not been done. Hence, in this study, the peripherl blood lymphocytes in brnd-nme nd generic tcrolimus were compred in stble liver trnsplnt recipients. Subjects nd Methods Ptients Sixty-four ptients underwent ABO-comptible liver trnsplnttion between 2 nd 213. All ptients were cytomeglovirus seropositive nd received right lobe grft from living donor. All recipients received tcrolimus s clcineurin inhibitor. The study ws pproved by the Smsung Medicl Center s Institutionl Review Bord in Seoul. The exclusion criteri were recipients of cyclosporine, sirolimus, or everolimus, s well s ptients with bile duct complictions, cytomeglovirus infection, biopsyproven cute rejection, heptocellulr crcinom recurrence, grft filure, or who died. Sixteen ptients were selected becuse they hd stble grft function without complictions. Ten ptients received brnd-nme tcrolimus (Progrf ; Astells Phrm, Tokyo, Jpn), while 6 ptients received generic tcrolimus (Tcrobell ; Chong Kun Dng Phrm, Seoul, Kore). One surgeon used brnd-nme tcrolimus nd the other surgeon used generic tcrolimus becuse they prescribed their fvorite mediction. All demogrphic nd clinicl dt were obtined from medicl records. All ptients were followed for the first 2 fter living donor liver trnsplnttion (LDLT). Immunosuppression The immunosuppressive protocol of our center ws used s described previously [1]. Bsiliximb (2 mg) ws used s n induction gent. All ptients were infused with prostglndin E 1, gbexte mesilte, nd methylprednisolone. Mintennce immunosuppressive therpy consisted of corticosteroids, tcrolimus, nd mycophenolte mofetil (MMF). Corticosteroids were withdrwn 3 months fter trnsplnttion. Peripherl Blood Lymphocyte Subpopultions Lymphocytes were nlyzed in fresh whole blood smples obtined preopertively nd,,, nd 2 fter liver trnsplnttion. For lymphocyte subset nlysis using multicolor flow cytometry, whole blood ws incubted with vrious monoclonl ntibodies specific for linege CD mrkers ccording to the mnufcturer s instructions [19]. All monoclonl ntibodies were purchsed from ebioscience (Sn Diego, CA, USA), BD Biosciences (Frnklin Lkes, NJ, USA), or Thermo Fisher Scientific (Rockford, IL, USA). The cocktil for γδt cells consisted of CD3- PerCP Cy5.5 (SK7, ebioscience), CD-APC-eFluor7 (RPA-T, ebioscience), CD-PE-Cy7 (SK1, ebioscience), TCR V delt 1-FITC (TS.2, Thermo Fisher Scientific), nd Vδ2 TCR-PE (B6, BD Biosciences). The cocktils for Tregs nd NK cells were s follows: CD-PerCP Cy5.5 (RPA-T, ebioscience), CD25-APC (BC96, ebioscience), nd FOXP3-PE (PCH11, ebioscience) for Tregs, nd CD3-PerCP Cy5.5 (SK7, ebioscience), CD16-APC- H7 (3G, BD Biosciences), nd CD56-FITC (MEM1, ebioscience) for NK cells. Briefly, 1 μl of whole blood contining different combintions of ntibodies ws incubted for 15 min t room temperture in the drk. This ws followed by red blood cell lysis nd wshing in phosphte-buffered sline (PBS). Cells were reconstituted with PBS contining.5% lbumin, nd FACS cquisition for cell surfce stining nlysis ws performed. For intrcellulr stining, surfce stined cells were fixed nd permebilized with Fix/Perm regent (ebioscience) nd then incubted with nti-foxp3 ntibody. Vrious lymphocyte subsets were defined s follows: CD3+CD+ for helper T cells, 222 Med Princ Prct 217;26: DOI: /5561 Kim/Kwon/Joh/Sinn/Choi/Prk/Kng/ Lee

3 Tble 1. Recipient chrcteristics of the brnd-nme nd generic tcrolimus groups in living donor liver trnsplnttion Brnd-nme tcrolimus (n = 1) Generic tcrolimus (n = 6) p vlue Bseline Sex (mle) 9 (9%) 5 (3.3%).696 Age, yers 56 ( 6) ( 67).36 Dignosis Alcoholic (%) 1 (16.7%) HBV 2 (2%) 1 (16.7%) HBV, HCC (%) 2 (33.3%) HCV, HCC (%) 1 (16.7%) NBNC (%) 1 (16.7%).176 History Hypertension 1 (1%) (%).2 Dibetes 2 (2%) 1 (16.7%).69 Child-Pugh clss.1 A 6 (6%) 3 (5%) B 2 (2%) 2 (33.3%) C 2 (2%) 1 (16.7%) MELD score (7 2) 9 (7 19).75 Periopertive Mcrostetosis, % 5 (1 5) 5 (5 1).313 GRWR.96 ( ) 1.25 ( ).7 Opertive time, min 532 (2 725) 539 (1 6).33 Cold ischemic time, min 76 (7 117) 95 (57 16).366 Wrm ischemic time, min 33 (2 5) 26 (1 55).5 Intensive cre unit sty fter trnsplnttion, n (dys) 6 ( 7) 6 (5 7).755 Hospitliztion, n (dys) 22 (21 2) 22 (17 2).755 Immunosuppression Tcrolimus concentrtion t 3 months 5.5 ( ) 5.3 (2.3.).7 MMF t 3 months 7 (7%) (66.7%).9 Steroids t 3 months 7 (7%) 5 (3.3%).551 Vlues re presented s n (%) or medins (rnge). HBV, heptitis B virus; HCC, heptocellulr crcinom; HCV, heptitis C virus; NBNC, non-b non-c; MELD, Model for End-Stge Liver Disese; GRWR, grft-torecipient weight rtio; MMF, mycophenolte mofetil. CD3+CD+ for cytotoxic T cells, CD3 CD56+CD16+ for NK cells, nd TCR Vδ1 nd Vδ2 for CD3+CD CD γδt cells. Cells were nlyzed on FACSCnto II using FACSDIVA softwre (BD Biosciences). Sttisticl Anlysis Ctegoricl vribles were compred using χ 2 test or Fisher exct test nd were expressed s percentges. Continuous vribles were compred using the Mnn-Whitney U test nd were expressed s medins nd rnges. Repeted mesures of lymphocyte popultions fter LDLT were nlyzed using mixed model. Absolute counts t ech point fter trnsplnttion were compred using the Mnn-Whitney U test with Bonferroni correction. Sttisticl significnce ws defined s p <.5. Dt nlysis ws performed using SPSS 21. (IBM Corp., Armonk, NY, USA). Results Bseline Chrcteristics The bseline chrcteristics of the ptients re summrized in Tble 1. There were no sttisticlly significnt differences in dignosis, gender, ge, history of hypertension, or dibetes, in Child-Pugh clss, or in Model for End-Stge Liver Disese (MELD) scores between the 2 groups. Opertive time, mcrostetosis, cold nd wrm ischemic time, grft-to-recipient weight rtio, sty in intensive cre unit fter LDLT, nd hospitliztion did not rech significnce in the periopertive period. The proportions of MMF nd steroids in the brnd-nme nd generic tcrolimus groups t 3 months fter LDLT were Brnd-Nme nd Generic Tcrolimus Med Princ Prct 217;26: DOI: /

4 2, 1,2 1, Color version vilble online CD3+ T cells (bsolute cell counts) 1,5 1, 5 CD+ T cells (bsolute cell counts) 6 2 p =.61 p =.31 2 b 2 1,5 CD+ T cells (bsolute cell counts) 1, 5 CD/CD p =.39 p =.117 c 2 d 2 Fig. 1. Chnges in αβt cells fter living donor liver trnsplnttion: CD3+ T cells ( ), CD+ T cells ( b ), CD+ T cells ( c ), nd CD/CD rtio ( d ). 22 Med Princ Prct 217;26: DOI: /5561 Kim/Kwon/Joh/Sinn/Choi/Prk/Kng/ Lee

5 2 Progrf Tcrobell p =.71 2 Progrf Tcrobell p =.177 Color version vilble online T cell (bsolute cell counts) * V 1 T cell (bsolute cell counts) b 2 15 Progrf 1. Progrf p =.79 Tcrobell Tcrobell. V 2 T cell (bsolute cell counts) 1 5 V 1/V p = c 2 d 2 Fig. 2. Chnges in γδt cells fter living donor liver trnsplnttion: γδt cells ( ), Vδ1 γδt cells ( b ), Vδ2 γδt cells ( c ), nd Vδ1/Vδ2 rtio ( d ). Brnd-Nme nd Generic Tcrolimus Med Princ Prct 217;26: DOI: /

6 CD3 CD56+ T cells (bsolute cell counts) 6 2 p =.326 CD+Foxp3+ T cells (bsolute cell counts) p =.26 * * Color version vilble online 2 b 2 Fig. 3. Chnges in CD3 CD56+ T cells ( ) nd CD+FoxP3+ T cells ( b ) fter living donor liver trnsplnttion. similr (7 vs. 66.7% for MMF nd 7 vs. 3.3% for steroids, respectively). In ddition, the concentrtion of tcrolimus in the brnd-nme nd generic tcrolimus groups ws 5.5 nd 5.3 ng/dl, respectively ( p =.) Peripherl Blood T-Cell Subsets between Brnd-Nme nd Generic Tcrolimus The bsolute lymphocyte counts nd bsolute cell counts of CD3+ T cells fter LDLT were incresed fter ( Fig. 1 ). The bsolute lymphocyte counts nd bsolute cell counts of CD3+ T cells in the brnd-nme tcrolimus group did not vry from those in the generic tcrolimus group. In subgroup nlysis, bsolute cell counts of CD+ T cells in the brnd-nme tcrolimus group were generlly higher thn in the generic tcrolimus group fter, but bsolute cell counts of CD+ T cells in the generic tcrolimus group were higher thn in the brnd-nme tcrolimus group ( Fig. 1 ). The CD/ CD rtio in the brnd-nme tcrolimus group ws higher thn in the generic tcrolimus group fter LDLT. Absolute cell counts of γδt cells in the brnd-nme tcrolimus group were higher thn in the generic tcrolimus group ( Fig. 2 ). Absolute cell counts of γδt cells in the generic tcrolimus group remined the sme fter LDLT, but bsolute cell counts of γδt cells in the brnd-nme tcrolimus group incresed fter. In subgroup nlysis, bsolute cell counts of Vδ1 nd Vδ2 γδt cells in the brnd-nme tcrolimus group were incresed fter. However, Vδ1/Vδ2 rtio in the generic tcrolimus group ws higher thn in the brnd-nme tcrolimus group ( Fig. 2 ). Absolute cell counts of CD3 CD56+ T cells in the brnd-nme tcrolimus group were higher thn in the generic tcrolimus group fter. Similrly, bsolute cell counts of CD+Foxp3+ T cells in the brndnme tcrolimus group were higher thn in the generic tcrolimus group fter ( Fig. 3 ). Discussion In this study, the levels of CD+Foxp3+ nd CD3 CD56+ T cells were higher in the brnd-nme tcrolimus group thn in the generic tcrolimus group fter 226 Med Princ Prct 217;26: DOI: /5561 Kim/Kwon/Joh/Sinn/Choi/Prk/Kng/ Lee

7 trnsplnttion. These findings indicte tht brnd-nme tcrolimus could hve more potentil s n immunosuppressnt thn generic tcrolimus becuse CD+Foxp3+ T cells re relted to grft tolernce. However, there were no sttisticlly significnt differences in CD3, CD, CD, or γδt cells between the brnd-nme nd the generic tcrolimus groups. Our study reveled tht the medin Vδ1/Vδ2 rtio in the generic tcrolimus group were more thn.2 t ech time, but medin Vδ1/Vδ2 rtio in the brnd-nme tcrolimus group ws below.2 t those times ( Fig. 2 ). The present study reveled tht the Vδ1/Vδ2 rtio in the generic tcrolimus group ws little higher thn in the brnd-nme tcrolimus group, but there ws no sttisticlly significnt difference between the 2 groups. Although there ws no sttisticlly significnt difference between the 2 groups, the present study demonstrted tht brnd-nme tcrolimus induced n increse in Vδ1 γδt cells fter compred to the generic tcrolimus group. γδt cells possess immunoregultory function; thus, incresed number of circulting γδt cells were observed in ptients with stble grft function [7, 1 16]. Tolernt liver recipients show n pprent ltertion in the peripherl blood γδt cell subsets, thus exhibiting n elevtion of Vδ1 γδt cells nd distinct increse of the Vδ1/Vδ2 rtio [1, 16, 2]. A recent study demonstrted n elevted Vδ1/Vδ2 rtio in tolernt peditric semillogeneic recipients s compred to mintennce immunosuppressive nd chronic rejection recipients [21]. Our findings reveled tht CD+Foxp3+ T cells in the brnd-nme tcrolimus group were higher thn in the generic tcrolimus group nd fter trnsplnttion. These observtions suggest tht brnd-nme tcrolimus might protect ginst liver llogrft rejection nd induce the effect of tolernce compred to generic tcrolimus. Tregs re well-chrcterized T CD+ cell subpopultion defined on the bsis of constitutive expression of high levels of IL-2 receptor-α chin (CD25), cytotoxic T lymphocyte-ssocited ntigen (CTLA-), nd forkhed/winged helix trnscription fctor P3 (FoxP3) [22, 23]. Tregs ply centrl role in the induction of immunologicl tolernce fter orgn trnsplnttion [22, 23]. Currently, immunosuppressive gents used in clinicl prctice potentilly ffect Treg cell popultions [22, 2]. Regultory pprovl for generic drugs is dependent on the demonstrtion of bioequivlence [3]. However, the phrmcokinetic properties of tcrolimus re ffected by number of ptient-relted vribles, such s gut function, concomitnt mediction, nd liver function [3]. Although generic tcrolimus my led to improved dherence to immunosuppression nd better helth outcomes for trnsplnt recipients, ptients nd helth cre providers should monitor tcrolimus trough levels more often if recipient is switched to generic tcrolimus [3, 25]. The present study hd severl limittions tht included (1) smll ptient popultion tht hd selection bis nd (2) the T effector cells were not nlyzed. The T effector cell/treg blnce is crucil for the initition nd progression of the grft rejection response. Therefore, n idel pproch to promote tolernce could be to deplete or minimize llorective T effector cells. (3) The T-cell chnges in the peripherl blood did not necessrily reflect their respective vlues in liver tissue. () Only bsolute cell counts in the T cell popultion were nlyzed. (5) There ws lck of ny evidence of moleculr-bsed dt; therefore, the mechnism of the effect of T cells by brnd-nme or generic tcrolimus could not be proved. Conclusions In this study the CD3 CD56+ nd CD+Foxp3+ T cells in the brnd-nme tcrolimus group were higher thn in the generic tcrolimus group in the peripherl blood T-cell popultions in stble liver trnsplnt recipients. This finding could indicte tht brnd-nme tcrolimus might hve more potentil immunosuppressive ctivity thn generic tcrolimus. Future studies monitoring the immune sttus of liver trnsplnt recipients in reltion to the type, number, nd function of T cells re recommended to elucidte these issues in clinicl liver trnsplnttion. Disclosure Sttement The uthors declre no conflicts of interest. References 1 Gu J, Wu X, Lu L, et l: Role of steroid minimiztion in the tcrolimus-bsed immunosuppressive regimen for liver trnsplnt recipients: systemtic review nd met-nlysis of prospective rndomized controlled trils. Heptol Int 21; : Li XC, Turk LA: An updte on regultory T cells in trnsplnt tolernce nd rejection. Nt Rev Nephrol 21; 6: Molnr AO, Fergusson D, Tsmplieros AK, et l: Generic immunosuppression in solid orgn trnsplnttion: systemtic review nd met-nlysis. BMJ 215; 35:h3163. Brnd-Nme nd Generic Tcrolimus Med Princ Prct 217;26: DOI: /

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