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The use of kinase inhibitors in solid organ transplantation
The use of kinase inhibitors in solid organ transplantation S Dholakia, JE Fildes, PJ Friend PII: DOI: Reference:
S0955-470X(17)30003-4 doi: 10.1016/j.trre.2017.02.008 YTRRE 450
To appear in:
Transplantation Reviews
Please cite this article as: Dholakia S, Fildes JE, Friend PJ, The use of kinase inhibitors in solid organ transplantation, Transplantation Reviews (2017), doi: 10.1016/j.trre.2017.02.008
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ACCEPTED MANUSCRIPT The use of kinase inhibitors in solid organ transplantation S Dholakia; JE Fildes; PJ Friend
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Nuffield Department of Surgical Science Oxford Transplant Centre Churchill Hospital Oxford OX3 7LE
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The Manchester Collaborative Centre for Inflammation Research (MCCIR) Institute of Inflammation and Repair Core Technology Facility University of Manchester Manchester M13 9NT
S Dholakia Email: [email protected] Mobile: +447787778649
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CORRESPONDING AUTHOR:
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CONFLICTS OF INTEREST AND SOURCE OF FUNDING: No funding was needed for the study and we declare no conflict of interest. CONTRIBUTIONS SD initiated the concept, SD, WC, collected and analysed results with SD writing the 1 st draft of the manuscript, all authors contributed equally to the script with JF and PF as the responsible consultants.
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Category of Article: Review – original work, has not been presented at any meeting or submitted elsewhere. We grant all rights of our work to your journal
ACCEPTED MANUSCRIPT The use of kinase inhibitors in solid organ transplantation S Dholakia; JE Fildes; PJ Friend
RI PT
Nuffield Department of Surgical Science Oxford Transplant Centre Churchill Hospital Oxford OX3 7LE
NU
SC
The Manchester Collaborative Centre for Inflammation Research (MCCIR) Institute of Inflammation and Repair Core Technology Facility University of Manchester Manchester M13 9NT
S Dholakia Email: [email protected] Mobile: +447787778649
MA
CORRESPONDING AUTHOR:
PT ED
CONFLICTS OF INTEREST AND SOURCE OF FUNDING: No funding was needed for the study and we declare no conflict of interest. CONTRIBUTIONS SD initiated the concept, SD, WC, collected and analysed results with SD writing the 1 st draft of the manuscript, all authors contributed equally to the script with JF and PF as the responsible consultants.
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Category of Article: Review – original work, has not been presented at any meeting or submitted elsewhere. We grant all rights of our work to your journal
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Abstract Introduction Despite the efficacy of current immunosuppression regimes used in solid organ transplantation, graft dysfunction, graft lost and antibody-mediated rejection, continue to be problematic. As a result; clear attraction in exploiting key potential targets controlled by kinase phosphorylation have led to a number of studies exploring the use of these drugs in transplantation. Aim In this review, we consider the role of tyrosine kinase as a target in transplantation and summarise the relevant studies on kinase inhibitors that have been reported to date. Method Narrative review of literature from inception to December 2016, using OVID interface searching EMBASE and MEDLINE databases. All studies related to kinase based immunosuppression were examined for clinical relevance with no exclusion criteria. Key ideas extracted and referenced. Conclusion The higher incidence of infections when using kinase inhibitors is an important consideration, however the number and range inhibitors and their clinical indications are likely to expand, but their exact role in transplantation is yet to be determined. ==========================================================
ACCEPTED MANUSCRIPT Introduction Clinical outcome following solid organ transplantation has significantly improved over the last two decades. However immunosuppressive regimes have unfortunately not evolved at the same pace, with the use of agents first described in the 1960s still being used today.
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Current immunosuppression protocols typically consist of a combination of a calcineurin inhibitor (cyclosporine or tacrolimus - the current cornerstone of immunosuppression), an inosine-5’-mono phosphate dehydrogenase inhibitor, and induction agents such as Alemtuzumab, Basiliximab, or Thymoglobulin.1,2,3
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When these agents were developed as immunosuppressants and adopted into clinical practice, our understanding of allorecognition and alloreactivity was comparatively limited. Consequently, immunosuppression dependent complications now represent one of the primary obstacles to successful transplantation, and this has provided the stimulus for the development of new immunosuppressive drugs of at least equivalent efficacy but improved safety profiles.
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In this review, we consider the role of the kinase family as a promising therapeutic target in transplantation as both as an anti-inflammatory and immunosuppressive agent, summarising relevant studies on kinase inhibitors that have been reported to date for use in solid organ transplantation. Kinase Overview
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There are 518 kinases in the human genome, divided into eight groups. Tyrosine kinases are one major subgroup of this large class of protein kinases; another important subgroup attaches the phosphate moiety to other amino acids (serine and threonine). Amongst the protein kinases, tyrosine kinases have attracted the greatest clinical attention so far, especially in oncology and rheumatology 8, 9
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The tyrosine kinase subfamily consists of two major groups. The receptor kinases (RK) contain an extracellular ligand-binding domain that is connected to the cytoplasmic catalytic domain by a transmembrane helix, and are activated by various growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).8,9,10
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Conversely, non-receptor cytoplasmic kinases (NRK) lack an extracellular component and are located in the cytosol, the nucleus and the inner surface of the plasma membrane. Major NRKs are those linked to sarcoma (SRC), Janus-associated kinase (JAK) and tyrosine kinase from oncogenic transcript that results from fusion of the ABL gene and breakpoint cluster region gene (BCR-ABL).10 The complex pathways exploited by these kinase inhibitors can be summarised here. (see figure one). Janus Kinase as a potential therapeutic target In the context of the immune response, the NRTK which has been studied most intensively to date is JAK, so named after the two-faced Roman god Janus, because JAK uniquely contains two almost identical phosphate-transferring domains but with two distinct and different functions; one domain exhibits the kinase activity while the other negatively regulates the kinase activity of the first.11 The JAK family in mammals consists of four members, namely JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2). The activation of JAK occurs by a ligand–receptor interaction that results in signalling through the phosphorylation of cytokine receptors and the creation of docking sites for a family of signalling proteins known as signal transducers and activators of transcription (STATs).
ACCEPTED MANUSCRIPT Phosphorylated STATs dimerise and then migrate to the nucleus where they become part of transcriptional regulatory machinery and lead to transcription of responsive genes. Under normal physiological conditions the two phosphate-transferring domains ensure that liganddependent activation of the JAK/STAT signalling is transient and tightly regulated. 12
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Signalling via cytokine and growth factor binding to their corresponding receptors on the target cell results in activation of JAKs, which phosphorylate and activate STAT pathways. In addition, STAT can be activated by SRC, ABL and EGFR. The importance of JAKs in vivo was first identified in knockout mice models and through demonstration of JAK3 mutations in patients with immunodeficiency. Mutation of JAK3 results in a severe combined immunodeficiency (SCID) characterised by an almost complete absence of T cells, NK cells and defective B cells.13
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In contrast with other ubiquitously expressed JAKs, JAK3 is primarily expressed in haematopoietically derived cells where it is associated with the IL-2 receptor common γ chain and mediates signalling by IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21; these being the cytokines that are critical for the development and maturation of T cells. The clinical phenotype selectively associated with JAK3 deficiency has led to the suggestion that targeting JAKs might be a strategy for the development of a new class of immunomodulatory drugs.13, 14
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Tofacitinib Formerly designated CP-690,550, tofacitinib was one of the first JAK inhibitors to enter the transplant arena. It was designed to inhibit JAK1 and JAK3 and, to a lesser extent, JAK2, having little effect on TYK2. As a result tofacitinib potently inhibits downstream signalling following common gamma chain cytokine receptor ligation, but also blocks signalling via IFN-γ, IL-6 and to a lesser extent, IL-12 and IL-23. Therefore tofacitinib can inhibit both innate and adaptive immune responses required for T cell differentiation and activation.15 The FDA has approved tofacitinib for use in Rheumatoid Arthritis in 2012 under the trade name of Xeljanz. However its use in transplantation has never been pursued despite data suggesting that tofacitinib may have therapeutic value.
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Preclinical Animal Studies: Using a murine heterotopic heart transplant model, treatment with tofacitinib resulted in a dosedependent increase in graft survival. Treatment was delivered for 28 days, and in the high dose groups the median survival times were greater than 60 days compared to 12 days survival for the placebo group. This study suggested that tofacitinib may represent an efficacious immunosuppressant following heart transplantation, albeit in mice . 17 The same group then performed a series of kidney transplants using mixed leukocyte reaction-mismatched, ABO blood group matched cynomolgus monkeys. Animals received either tofacitinib (n = 18) or placebo (n = 3 -one animal died and so was excluded) and were killed at day 90 or earlier in cases of allograft rejection. The mean survival time in animals treated with tofacitinib was significantly longer than that in control animals (53 ± 7 days vs. 7 ± 1 days, P <0.0003) and was positively correlated with drug exposure. However it is important to note that the control group had longer cold ischaemic times and higher rates of MHC mismatch. Four treated animals killed at 90 days were found to have normal renal function and low-grade rejection on final pathology. The occurrence of rejection was significantly delayed in treated animals but was not prevented (46 ± 7 days from transplantation vs. 7 ± 1 days in controls, P <0.0003). This study confirmed the immunosuppressive potential of tofacitinib but showed independently it was unable to prevent rejection and had significant dose dependent side effects.18 Building on this, further pharmacodynamic assessments identified in vitro exposure to tofacitinib resulted in a significant reduction of IL-2- enhanced IFNγ production by T cells, reduced T-cell surface expression of CD25 and CD71, and reduced T-cell proliferative capacities. These results mirrored similar data in animals dosed with tofacitinib, which displayed a significant reduction of natural killer cell and T-cell numbers whereas CD8+ effector memory T-cell populations remained unaffected.19
ACCEPTED MANUSCRIPT The combination of tofacitinib with mycophenolate mofetil (MMF) significantly improved allograft survival, with the mean survival time in animals treated with MMF alone being significantly extended in animals that concurrently received tofacitinib (23 ± 1 days vs. 59.5 ± 9.8 days, P <0.02). 20
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The side effects observed during these studies during high dose administration of tofacitinib included anaemia and polyoma virus nephritis, yet these were not seen when combined with MMF, suggesting a safe and effective combination may have been identified. As tofacitinib has pan JAK activity; inhibition of JAK2 that mediates erythropoiesis is thought to be a contributing factor to the anaemia seen with its use. 18,19,20
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Phase 1 trial The results of the animal studies enabled the initiation of a dose-escalation study in human renal allograft recipients. The safety effects and tolerability on lymphocyte subsets was a key focus as well as assessing the pharmacokinetics of tofacitinib when co-administered with MMF. 28 patients were recruited in 2008; where six received tofacitinib 5 mg twice daily, six patients received 15 mg, 10 patients received 30 mg, and six patients received placebo. 21 The most common adverse events observed in the study included infection and gastrointestinal symptoms (abdominal pain, diarrhoea, dyspepsia, and vomiting). Higher doses of tofacitinib were associated with anaemia and decreased natural killer cell counts (50%), mirroring what had been observed in earlier primate studies, yet numbers of neutrophils, platelets and T helper cells remained unchanged.21
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The effect of tofacitinib following 29 days of 30 mg administration was investigated in eight kidney transplant recipients. In this study key circulating cytokines within the JAK/STAT pathway were reduced compared to the placebo group . IL-2-induced phosphorylated STAT5 expression in YT cells (immortalised human NK ‘like’ cells) was reduced in the presence of serum samples collected from whole blood on day 29 compared with those collected at baseline. Phosphospecific flow cytometry also revealed tofacitinib reduced IL-2-induced phosphorylated STAT5 in CD3+, CD3+CD4+ and CD3+CD8+ subpopulations as well as interferon gamma (IFNγ)
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production by mononuclear cells. However, examination of blood at day 29 again showed persistent anaemia following the administration of tofacitinib, which seemed to be an ongoing problem.22, 23
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Phase 2a trial Despite the increased incidence of anaemia, a randomized pilot study comparing tofacitinib (15 mg and 30 mg, n = 20 in each group) vs tacrolimus (n = 21) in de novo renal allograft recipients was initiated in 2009. Patients additionally received an IL-2 receptor antagonist, MMF and corticosteroids, which are all part of current standard treatment regimes in kidney transplantation.24 Tofacitinib doses were reduced following 6 months of therapy due to high incidence of BK virus nephropathy in patients treated with high dose tofacitinib, and as a result MMF was discontinued in this group. The 6-month biopsy-proven acute rejection rates were one out of 20, four out of 20 and one out of 21 for 15 mg tofacitinib, 30 mg tofacitinib and tacrolimus,groups respectively, demonstrating that low dose tofacitinib had equivalent efficacy to tacrolimus, but was significantly associated with clinically relevant infections at higher doses. 24 BK virus nephropathy developed in four out of 20 patients in the 30 mg twice-daily administed tofacitinib group. The 6-month rates of cytomegalovirus disease were 2 out of 20, 4 out of 20 and 0 out of 21 for the 15 mg, 30mg tofacitinib groups and tacrolimus groups, respectively. 24 The estimated glomerular filtration rate measured was >70 ml/minute at 6 and 12 months for all groups. In the tofacitinib arms, there were modest lipid elevations and a trend toward more frequent anaemia and neutropenia during the first 6 months.25, 26 This data suggested that co-administration of 30 mg BD tofacitinib with MMF was clearly associated with over immunosuppression. At the 15 mg twice-daily dose, the efficacy and safety profile produced equivocal results to the current standard, tacrolimus yet the incidence of infection was higher and more significant as tofacitinib doses were escalated.24
ACCEPTED MANUSCRIPT Phase 2b trial A phase 2b, randomised, multi-centre, partially blinded study was designed to compare a low or high intensity regime of tofacitinib vs cyclosporine in 331 low to moderate risk de-novo kidney transplant patients. All patients also received basiliximab induction, MMF and corticosteroids.
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Two primary non inferiority endpoints were identified as; incidence of biopsy-proven acute rejection at 6 months or biopsy proven rejection meeting serum creatinine criteria at month 6 (serum creatinine increase ≥0.3 mg/dl and ≥20% from pre-rejection baseline) and measured glomerular filtration rate at month 12. Clinical biopsy-proven acute rejection (11%, 7% and 9% respectively) was observed for the high intensity, low intensity and cyclosporine groups, there was no statistical difference between these groups for rejection. Measured glomerular filtration rates were higher at month 12 for both tofacitinib groups versus the cyclosporine group (65 ml/minute, 65 ml/ minute vs. 54 ml/minute) p<0.01. Fewer patients in the tofacitinib arms developed chronic allograft nephropathy at month 12 compared with cyclosporine patients (25%, 24% vs. 48%). Serious infections, particularly cytomegalovirus and BK virus, developed in 45%, 37% and 25% with high dose tofacitinib again showing higher rates of infection, while cyclosporine had the lowest incidence. Additionally, anaemia, neutropenia and post-transplant lympho-proliferative disorder (PTLD) occurred in four patients in the tofacitinib arm. 27
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The authors concluded that tofacitinib remained equivalent to cyclosporine in preventing acute rejection, and is associated with improved renal function and less chronic allograft histological injury, however the high incidence of infection and anaemia which has been a persistent issue, although dose dependent, ultimately led to the termination of in the agents use in transplantation.27, 28
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Spleen Tyrosine Kinase Spleen tyrosine kinase (SYK), along with Zap-70, is a member of the Syk family of tyrosine kinases. These non-receptor cytoplasmic tyrosine kinases share a characteristic dual SH2 domain separated by a linker domain. SYK has a well-characterised role in the intracellular signalling cascade for classic immunoreceptors such as Fc receptors (FcRs) and the B-cell receptor.8 While Syk and Zap-70 are primarily expressed in hematopoietic tissues, there is expression of Syk in a variety of other tissues making them an attractive target within the field of transplantation. Like JAK3, Zap70 deficiency can also cause severe combined immunodeficiency (SCID), but in this case there is preferential loss of CD8+ T cells. For this reason, Zap70 also remains a target but unfortunately, a clinically useful compound has not yet emerged for testing in the transplant setting.29, 30
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Kidney allografts induce strong T-cell and antibody mediated responses which are known to orchestrate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leukocytes excluding mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and several integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but its pharmacological inhibition reducing activation of monocytes/macrophages and neutrophils makes it an attractive target as an immunosuppressant.30, 31 Fostamatinib is an oral pro-drug and potent inhibitor of immunoglobulin E (IgE) and IgG mediated activation of Fc receptor signalling, with its primary target being spleen tyrosine kinase (Syk). It has been used in rheumatoid arthritis and the treatment of a number of leukaemias. 8, 9 Smith et administered Fostamatinib from day 4 to 10 to Wistar Kyoto rats, and reported reduced glomerular crescents by 100% (P < 0.01) compared with controls. When administered from days 7 to 14, the established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function improved when compared to controls (P < 0.001).32 Hence treatment of Fostamatinib reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, as well as reduced renal monocyte chemo-attractant protein-1 (MCP-1) and IL-1β.32, 33 Pamuk et al, demonstrated that fostamatinib is protective against ischaemia reperfusion in a murine model. Mice were fed with food containing fostamatinib (3 g/kg low dose, 5 g/kg high dose or control, n= 6 per group) for 6 days before induction of intestinal ischaemia reperfusion.
ACCEPTED MANUSCRIPT Artificial ischaemia was performed via anaesthesia and clamping of vessels for a period of 30minutes, which in the treatment groups resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local intestinal and remote lung injury and the beneficial effect was associated with reduced IgM and complement 3 depositions in the tissues, and significant reduction of polymorphonuclear cell infiltration. 34
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The Syk inhibitor CC0482417 (Celgene) was reported to reduce renal allograft rejection and histological damage in the rat model. Recipient rats underwent kidney transplantation and were treated with CC0482417, 30 mg/kg twice daily or placebo, from 1 h before surgery until being euthanased 5 days later. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries.35
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Fostamatinib is currently undergoing a phase 1 trial for chronic graft vs. host disease development following allogeneic stem cell transplantation, but results are yet to be published. 36
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Sotrastaurin Sotrastaurin (AEB071) represents a new class of immunosuppressant, an oral inhibitor of protein kinase C (PKC)F, PKCa, and PKCb, which are essential for T and B cell activation and proliferation. In rodents and primates, sotrastaurin prevents allograft rejection and reduces inflammation, but perhaps more importantly has a reduced side effect profile with no evidence of renal failure, diabetes or hypertension.37
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In the first reported clinical trial, de novo renal transplant recipients with immediate graft function were randomized 1:2 to tacrolimus (control,n=44) or sotrastaurin (300 mg b.i.d.; n=81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary endpoint was a composite of treated biopsy-proven acute rejection (BPAR), graft loss, death or lost to follow-up at month 3. The main safety assessment was estimated glomerular filtration rate (eGFR) and modification of diet in renal disease (MDRD) at month 3. Composite efficacy failure at month 3 was higher in the sotrastaurin group compared to control (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p =0.003), which led to early study termination. Median (± standard deviation [SD]) eGFR was higher for sotrastaurin versus control at all time points from day 7 (month 3: 59.0 ± 22.3 vs. 49.5 ± 17.7 mL/min/1.73m2) p = 0.006. The most common adverse events were gastrointestinal disorders (control: 63.6%; sotrastaurin: 88.9%), which led to study-medication discontinuation in two sotrastaurin patients. The study reported a lower degree of efficacy but better renal function with the calcineurin-inhibitor-free regimen of sotrastaurin + MPA versus the tacrolimus-based control.37 Tedesco-Silva et al followed this with a phase two human de novo renal transplant study, which identified recipients with immediate graft function who were randomized 1:2 to tacrolimus (control, n=44) or sotrastaurin (300 mg b.i.d.; n=81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary composite endpoints were treated biopsyproven acute rejection (BPAR), graft loss, death or lost to follow-up at month three. Composite efficacy failure at month three was found to be higher for the sotrastaurin versus control regimen (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p = 0.003), which led to early study termination.38,39 This was followed by an efficacy and safety study of sotrastaurin with tacrolimus to assess dose ranging, in a non-inferiority study in renal transplant recipients. A total of 298 patients were randomized 1:1:1:1 to receive sotrastaurin 100mg BD, sotrastaurin (200 mg BD) + standardexposure tacrolimus (SET) or reduced-exposure tacrolimus (RET) (SET: n = 76; RET: n = 66), or control (SET + mycophenolic acid [MPA, 720 mg BD]; n = 73). All patients received basiliximab and corticosteroids.40, Composite efficacy failure rates at month 12 were 18.8%, 12.4%, 10.9% and 14.0% for the sotrastaurin 100, 200 and 300 mg, and MPA groups, respectively. The median estimated
ACCEPTED MANUSCRIPT glomerular filtration rates were 55.7, 53.3, 64.9 and 59.2 mL/min, respectively. Patients treated with sotrastaurin developed more frequent tachycardia, and were more frequently discontinued due to adverse side effects.39,40
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Further to this, sotrastaurin with tacrolimus was assessed in a 24-month, multicentre, phase II study in de novo liver transplant recipients. A total of 204 patients were randomized (1:1:1:1) to Sotrastaurin 200 mg B.D. + standard-exposure TAC (n = 50) or reduced-exposure TAC (n = 52), Sotrastaurin 300 mg b.i.d. + reduced-exposure TAC (n = 50), or mycophenolate mofetil (MMF) 1 g b.i.d. + standard-exposure TAC (control, n = 52); all with steroids.41
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Owing to premature study termination, treatment comparisons were only conducted at six months, where composite efficacy failure rates were 25.0%, 16.5%, 20.9% and 15.9% respectively showing sotrastaurin groups having a higher incidence of rejection and adverse events.41
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As a result, development of this compound has been halted due to the continued premature study discontinuation, higher incidences of acute rejection, graft loss, and adverse side effects when compared to standard immunosuppression, deeming it an efficacy failure. 39,40 Despite the promise shown by protein kinase C inhibitors, it is clear that T cell activation is not significantly controlled using this mechanism and so the inhibition of this pathway appears to have little clinical benefit.
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Sunitinib Sunitinib is a RTK for gastrointestinal stromal tumour and metastatic renal cell carcinoma. It inhibits PDGF receptors PDGFR-α and -β and VEGF receptors VEGFR-1, −2, −3 at similar concentrations.8
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Sunitinib has been successfully used in the treatment of imatinib-resistant tumours, which may suggest better efficacy. Is has also been shown to be better than imatinib in preventing PDGFR-β phosphorylation. Rintala et al have previously reported that in an experimental kidney transplantation model, inhibition of PDGF with imatinib prevents chronic rejection. They also found Sunitinib to decrease neointimal formation and smooth muscle cell proliferation in a dose dependent manner in the rat kidney model where grafts preserved better renal function. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expression demonstrating that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation.
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Despite these positive results, it is important to note there is a risk of nephrotic syndrome developing with use of this compound and Sunitinib is yet to be trialled in the context of human transplantation, hence the lack of other studies in this area, suggesting possibility of further work42, 43 Kinase Inhibitors in Ischaemia Reperfusion The various mechanisms participating in the development of ischemia reperfusion injury have been extensively reviewed elsewhere. However the result of a prolonged oxygen deprivation promoting tissue hypoxia causes ATP cell depletion, cell swelling and mitochondrial disruption. The inflammatory cascade is complex, but what is evident is the ubiquitous presence of kinasecontrolled mechanisms. This has identified several kinase family members as potential pharmacological targets for the treatment of ischemia reperfusion injury. Much of the work done in this area is experimental using animal models, which we have summarised below. Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported, where activation of two calcium-insensitive isoforms of the novel PKC subfamily, delta and epsilon, play opposing roles in ischaemia/reperfusion injury. Activation of delta PKC during reperfusion induces cell death through the regulation of mitochondrial function and induction of apoptosis and oncosis. In contrast, activation of epsilon PKC before ischaemia protects mitochondrial function and diminishes apoptosis and oncosis. Both the delta-PKC inhibitor and epsilon-PKC activator conferred cardioprotection against ischemia/reperfusion injury in transgenic mice expressing these peptides in the heart, and co-expression of both peptides
ACCEPTED MANUSCRIPT exerted greater cardioprotective effects than that obtained by the expression of each peptide alone.44
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Using a rat heart transplant model, Tanaka et al identified that combined treatment with protein kinase C activator and delta-protein kinase C inhibitor reduced ischemia-reperfusion injury and decreased the resulting graft coronary artery disease induced by prolonged ischemia. The group analysed the extent of ischemia-reperfusion injury by measuring superoxide generation, myeloperoxidase activity, tumour necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production, and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and caspase 2, 3, 8, and 9 activity.45
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Outside the field of transplantation, work within myocardial ischaemia has identified the inhibition of Rho-kinase activation in leukocytes. Studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury, are cardio protective and reduce cytokine production, but the role of Rho-kinase in leukocytes during I/R injury is not well understood. 46,47 Kitano et al used mice which were subjected to 30-min ischaemia and reperfusion injury. It was found Rhokinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumour necrosis factor (TNF)-α in leukocytes, compared with saline as the vehicle.48
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During liver transplantation, nonalcoholic steatohepatitis (NASH) aggravates the ischemia reperfusion injury by activating various kinases and subsequently releasing cytokines and chemokines. Yang et al, have identified that sorafenib protects NASH rats from IR injury by interfering with the inflammation, necrosis, and apoptotic responses which cause leukocytedependent hepatic microcirculatory dysfunction. The hepatoprotective effects of sorafenib seem to work through the inhibition of the Rho-kinase-dependent Raf/MEK/ERK pathway, which is up regulated during IR injury in the livers of NASH rats.49
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In animal models of lung ischemia–reperfusion and acute inflammatory lung injury, mitogenactivated protein kinases (p38, c-jun N-terminal kinase, extracellular signal–regulated kinase) are activated after oxidative stress. Wolf et al has shown using an experimental rat model that the inhibition of p38 and c-jun kinase provided significant protection from injury.50 Similar animal studies have been performed in liver transplant rat models where Sotraustaurin prevented T lymphocyte activation via a calcineurin-independent pathway, which is of particular appeal as it has also been documented that the role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI) is critical. 51 This study explored the cytoprotective functions of sotraustaurin in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from SD rats were stored for 30h at 4° in UW solution, and then transplanted to syngeneic recipients. Sotraustaurin treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. Sotraustaurin treatment decreased activation of T cells and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished NF-κB/ERK signalling, depressed pro-apoptotic cleaved caspase-3, yet upregulated anti-apoptotic Bcl-2/Bclxl and hepatic cell proliferation. 51 Young Dong et al, who investigated the use of Nafamostat mesilate which inhibits various serine proteases and mitogen activate protein kinase (MAPK) generated during inflammation, has recently made the leap from these experimental animal studies to a single arm pilot study. Nafamostat meslilate is widely used in Asia as an anti-inflammatory and anticoagulant agent. In this study, 10 human livers were flushed with a solution consisting of nafamostat mesilate following graft perfusion using standard HTK solution; these then were then transplanted into 10 consenting patients. Most patients received an emergency deceased donor liver, with mean cold and warm ischemic times of 373 (+/-) and 35 (+/-) minutes, respectively. Due to the limited sample size, a relevant statistical analysis was not possible. However the authors observed decreased mean aspartate aminotransferase (AST) and alanine aminotransferase
ACCEPTED MANUSCRIPT (ALT) levels on the first and third postoperative day as well as lower peak AST and ALT levels, suggesting the impact of ischemia reperfusion had been reduced. 52 Conclusion
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The use of kinase inhibitors has expanded well beyond malignancies to encompass autoimmune disease and many other applications. The role of kinase inhibitors in transplantation remains precarious; but despite their variable safety profile and adverse side effects, protein kinase inhibitors continue to have a role in immune disorders, but the initial use as a potential immunosuppressant in transplantation has shown to be troubled. However, optimising the dose and duration of treatment needed to achieve optimal immunosuppression combined with improved safety may allow this class of medication to be reconsidered in the future.
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Perhaps more importantly, the use of kinase inhibitors may be of benefit in the amelioration of ischaemia reperfusion rather than long-term immunosuppression, capitalising on the beneficial effects of widespread effect without the issues faced of infection associated with longer-term use.
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The number of kinase inhibitors and the range of their clinical indications are likely to expand dramatically in the immediate future. Precisely how these drugs are used in combination with or in place of other therapies such as biologics, steroids and many others remains to be determined. References
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Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clinc Invest 2000, 105:3-7
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Leonard WJ: Role of Jak kinases and STATs in cytokine signal transduction. Int J Hematol 2001, 73:271-7
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Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 241 (4861):42-52
10. Dengjel J, Kratchmarova I, Blagoev B. Receptor tyrosine kinase signalling: a view from quantitative proteomics. Mol Biosyst 2009; 5(10):1112-21
ACCEPTED MANUSCRIPT 11. Wiley HS, Burke PM. Regulation of receptor tyrosine kinase signalling by endocytic trafficking. Traffic 2001; 2(1):12-8
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12. Wilks AF, Harpur AG, Kurban RR, Ralph SJ, Zürcher G, Ziemiecki A: Two-novel proteintyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol Cell Biol 1991, 11:2057-2065. 13. Coppo P, Flamant S, De Mas V, Jarrier P, Guillier M, Bonnet ML, Lacout C, Guilhot F, Vainchenker W, Turhan AG. BCR-ABL activates STAT3 via JAK and MEK pathways in human cells. Br J Haematol 2006; 134(2):171-9
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14. Wilson LJ: Recent patents in the discovery of small molecule inhibitors of JAk3. Expert Opin Ther Pat 2010, 20:609-623. 15. O’Shea JJ, Pesu M, Borie DC, Changelian PS: A new modality for immunosuppression: targeting the JAK/STAT pathway. Nat Rev Drug Discov 2004, 3:555-564.
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16. Ghoreschi K, Jesson MI, Li X, Lee JL, Ghosh S, Alsup JW, Warner JD, Tanaka M, StewardTharp SM, Gadina M, Thomas CJ, Minnerly JC, Storer CE, LeBranche TP, Radi Za, Dowty ME. Head RD, Meyer DM, Kishore N, Oshea J. Modulation of inate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol 2011; 186:4234-43
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17. Changelian PS, Flanagan ME, Ball DJ, Kent CR, Magnuson KS, Martin WH, Rizzuti BJ, Sawyer PS, Perry BD, Brissette WH, McCurdy SP, Kudlacz EM, Conklyn MJ, Elliott EA, Koslov ER, Fisher MB, Strelevitz TJ, Yoon K, Whipple DA, Sun J, Munchhof MJ, Doty JL, Casavant JM, Blumenkopf TA, Hines M, Brown MF, Lillie BM, Subramanyam C, Shang-Poa C, Milici AJ, et al.: Prevention of organ allograft rejection by a specific Janus Kinase 3 inhibitor. Science 2003, 302:875-878.
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18. Borie DC, Changelian PS, Larson MJ, Si MS, Paniagua R, Higgins JP, Holm B, Campbell A, Lau M, Zhang S, Flores MG, Rousvoal G, Hawkins J, Ball DA, Kudlacz EM, Brissette WH, Elliott EA, Reitz BA, Morris RE: Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates. Transplantation 2005, 79:791-801.
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19. Paniagua R, Si MS, Flores MG, Rousvoal G, Zhang S, Aalami O, Campbell A, Changelian PS, Reitz BA, Borie DC: Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allograft. Transplantation 2005, 80:1283-1292. 20. Borie DC, Larson MJ, Flores MG, Campbell A, Rousvoal G, Zhang S, Higgins JP, Ball DJ, Kudlacz EM, Brissette WH, Elliott EA, Reitz BA, Changelian PS: Combined use of the JAJ3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates. Transplantation 2005, 80:1756-1764. 21. Van Gurp E, Weimar W, Gaston R, Brennan D, Mendez R, Pirsch J, Swan S, Pescovitz MD, Ni G, Wang C, Krishnaswami S, Chow V, Chan G: Phase 1 dose escalation study of CP690,550 in stable renal allograft recipients: preliminary findings of safety, tolerability, effects on lymphocyte subsets and pharmacokinetics. Am J Transplant 2008, 8:17111718. 22. Quaedackers ME, Mol W, Korevaar SS, van Gurp EA, vanIjcken WF, Chan G, Weimar W, Baan CC: Monitoring of the immune modulatory effect of CP-690,550 by analysis of the JAK/STAT pathway in kidney transplant patients. Transplantation 2009, 88:1002-1009. 23. Van Gurp E, Schoordijk-Verschoor W, Klepper M, Korevaar SS, Chan G, Weimar W, Baan CC: The eff ect of the JAK inhibitor CP-690,550 on peripheral immune parameters in stable kidney allograft patients. Transplantation 2009, 87:79-86.
ACCEPTED MANUSCRIPT 24. Busque S, Leventhal J, Brennan DC, Steinberg S, Klintmalm G, Shah T, Mulgaonkar S, Bromberg JS,Vincenti F, Hariharan S, Slakey D, Peddi VR, Fisher RA, Lawendy N, Wang C, Chan G: Calcineurin-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients. Am J Transplant 2009, 9:1936-1920.
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25. Wojciechowski D, Vincenti F: Targeting JAK3 in kidney transplantation: current status and future options. Curr Opin Organ Transplant 2011, 16:614-619. 26. Sewgobind VD, Quaedackers ME, van der Laan LJ, Kraaijeveld R, Korevaar SS, Chan G, Weimar W, Baan CC: The jak inhibitor CP-690,550 preserves the function of CD4CD25FoxP3 regulatory T cells and inhibits effector T cells. Am J Transplant 2010, 10:1785-1795.
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27. Vincenti F, Tedesco-Silva H, Busque S, O’Connell P, Friedewald J, Cibrik D, Budde K, Yoshida A, Cohney S, Weimar W, Kim YS, Lawendy N, Lan SP, Kudlacz E, Krishnaswami S, Chan G: Randomized phase 2b trial of tofacitinib (CP-690,550) in de novo kidney transplant patients: effi cacy, renal function and safety at one year. Am J Transplant 2012, 12:2446-2456.
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28. Borie DC, O’Shea JJ, Changelian PC: JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants. Trends Mol Med 2004, 10:532-541. 29. Chan AC, van Oers NS, Tran A. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J. Immunol.1994;152:4758–4766.
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30. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB. Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature. 1995;378:303–306. 31. Braselmann S, Taylor V, Zhao H. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J. Pharmacol. Exp. Ther. 2006;319:998–1008.
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32. Smith J, McDaid J, Bhangal G, Chawanasuntorapoj R, Masuda E. Terence Cook H, Pusey C, Tam F. A spleen tyrosine kinase inhibitor reduces the severity of established glomerulonephritis. J Am Soc Nephrol. 2010; 21(2): 231-236.
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33. Bahjat FR, Pine PR, Reitsma A. An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus. Arthritis Rheum. 2008;58:1433–1444. 34. Pamuk O, Lapchak P, Rani P, Pine P, Lucca J, Tsokos G. Spleen tyrosine kinase inhibition prevents tissue damage after ischaema-reperfusion. Am J Physiol Gastrointest Liver Physiol. 2010; 299(2):G391-G399. 35. Chandran S, Tesch G, Han Y, Woodman N, Mulley W, Kanellis J, Blease K, Ma F, NikolicPaterson D. Spleen tyrosine kinase contributes to acute renal allograft rejection in the rat. Int J Exp Pathol. 2015; 96(1):54-62 36. https://clinicaltrials.gov/ct2/show/NCT02611063 37. Trotter JF, Levy G. Sotrastaurin in Liver Transplantion: Has it had a fair trial. Am J Transpl 2015; 15:1137-1138 38. Friman S, Arns W, Nashan B, Vincenti F, Banas B, Budde K, Cibrik D, Chan L, Klempnauer J, Mulgaonkar S, Nicholson M, Wahlberg J, Wissing K-M, Abrams K, Witte S, Woodle ES. Sotrastaurin, a novel small molecule inhibiting protein-kinase C: randomized phase II study in renal transplant recipients. Am J Transplant. 2011;11:1444–55
ACCEPTED MANUSCRIPT 39. Tedesco-Silva H, Kho MM, Hartmann A, Vitko S, Russ G, Rostaing L, Budde K et al. Sotrastaurin in calcineurin inhibitor-free regimen using everolimus in de novo kidney transplant recipients. Am J Transpl 2013; 7:1757-1768
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40. Russ GR, Tedesco-Silva H, Kuypers DR, Cohney S, Langer RM, Witske O, Eris J et al. Efficacy of sotrastaurin plus tacrolimus after de novo kidney transplantation: randomized, phase II trial results. Am J Transpl 2013; 13(7):1746-56 41. Pascher AA, De Simone PB, Pratschke J. Protein kinase C inhibitor sotrastaurin in de novo liver transplant recipients: A randomized phase II trial. Am J Transpl 2015;15(5):1283-92
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42. Rintala JM, Savikko J, Palin N, Rintala SE, Koskinen PK, Von Willebrand E. Oral platelet derived growth factor and vascular endothelial growth factor inhibitor sunitinib prevents chronic allograft injury in experimental transplantation model. Transplantation 2016; 100(1):103-10
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43. Takahashi D, Nagahama K, Tsuura Y, Tanaka H, Tamura T. Sunitinib-induced nephrotic syndrome and irreversible renal dysfunction. Clin Exp Nephrol 2012;16(2):310-5
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44. Inagaki K, Hahn HS, Dorn GW 2nd, Mochly-Rosen D. Additive protection of the ischemic heart ex vivo by combined treatment with delta-protein kinase C inhibitor and epsilonprotein kinase C activator. Circulation, 2003; 108(7):869-75
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45. Tanaka M, Gunawan F, Terry R, Inagaki K et al. Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators. J Thorac Cardiovasc Surg. 2005; 129(5):1160-7 46. Hamid SA, Bower HS, Baxter GF. Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am J Physiol Heart Circ Physiol. 2007; 292: H2598–2606.
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47. Bao W, Hu E, Tao L, Boyce R, Mirabile R et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res. 2004; 61(3):548-58
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48. Kitano K, Usui S, Ootsuji H, Takashima S-i, Kobayashi D, Murai H, et al. Rho-Kinase Activation in Leukocytes Plays a Pivotal Role in Myocardial Ischemia/Reperfusion Injury. PLoS ONE. 2014; 9(3): e92242. 49. Yang Y, Huang YT, Lee TY, Chan CC et al. Rho-kinase-dependent pathway mediates the hepatoprotective effects of sorafenib against ischaemia/reperfusion liver injury in rats with non-alcoholic steatohepatitis 50. Wolf PS, Merry HE, Farivar AS, McCourtie AS, Mulligan MS. Stress-activated protein inhibition to ameliorate lung ischemia reperfusion injury. J Thorac Cardiovasc Surg. 2008 Apr; 135(4):856 51. Kamo N, Shen X, Ke B, Busuttil R, Kupiec-Weglinski J. Sotrastaurin a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthoptic liver transplantation. Am K Transplant 2011; 11(11): 2499-2507 52. Young-Dong Y, Dong-Sik K, Sung-Won J, Jae-Hyun H. The effect of nadamostat mesilate to minimise ischemic reperfusion injury of the liver following transplantation: a single arm pilot study. TTS abstract 2016. http://www.tts.org/component/tts/?view=presentation&id=182025
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Figure 1
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ACCEPTED MANUSCRIPT
S0955-470X(17)30003-4 doi: 10.1016/j.trre.2017.02.008 YTRRE 450
To appear in:
Transplantation Reviews
Please cite this article as: Dholakia S, Fildes JE, Friend PJ, The use of kinase inhibitors in solid organ transplantation, Transplantation Reviews (2017), doi: 10.1016/j.trre.2017.02.008
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ACCEPTED MANUSCRIPT The use of kinase inhibitors in solid organ transplantation S Dholakia; JE Fildes; PJ Friend
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Nuffield Department of Surgical Science Oxford Transplant Centre Churchill Hospital Oxford OX3 7LE
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The Manchester Collaborative Centre for Inflammation Research (MCCIR) Institute of Inflammation and Repair Core Technology Facility University of Manchester Manchester M13 9NT
S Dholakia Email: [email protected] Mobile: +447787778649
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CORRESPONDING AUTHOR:
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CONFLICTS OF INTEREST AND SOURCE OF FUNDING: No funding was needed for the study and we declare no conflict of interest. CONTRIBUTIONS SD initiated the concept, SD, WC, collected and analysed results with SD writing the 1 st draft of the manuscript, all authors contributed equally to the script with JF and PF as the responsible consultants.
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Category of Article: Review – original work, has not been presented at any meeting or submitted elsewhere. We grant all rights of our work to your journal
ACCEPTED MANUSCRIPT The use of kinase inhibitors in solid organ transplantation S Dholakia; JE Fildes; PJ Friend
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Nuffield Department of Surgical Science Oxford Transplant Centre Churchill Hospital Oxford OX3 7LE
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The Manchester Collaborative Centre for Inflammation Research (MCCIR) Institute of Inflammation and Repair Core Technology Facility University of Manchester Manchester M13 9NT
S Dholakia Email: [email protected] Mobile: +447787778649
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CORRESPONDING AUTHOR:
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CONFLICTS OF INTEREST AND SOURCE OF FUNDING: No funding was needed for the study and we declare no conflict of interest. CONTRIBUTIONS SD initiated the concept, SD, WC, collected and analysed results with SD writing the 1 st draft of the manuscript, all authors contributed equally to the script with JF and PF as the responsible consultants.
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Category of Article: Review – original work, has not been presented at any meeting or submitted elsewhere. We grant all rights of our work to your journal
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Abstract Introduction Despite the efficacy of current immunosuppression regimes used in solid organ transplantation, graft dysfunction, graft lost and antibody-mediated rejection, continue to be problematic. As a result; clear attraction in exploiting key potential targets controlled by kinase phosphorylation have led to a number of studies exploring the use of these drugs in transplantation. Aim In this review, we consider the role of tyrosine kinase as a target in transplantation and summarise the relevant studies on kinase inhibitors that have been reported to date. Method Narrative review of literature from inception to December 2016, using OVID interface searching EMBASE and MEDLINE databases. All studies related to kinase based immunosuppression were examined for clinical relevance with no exclusion criteria. Key ideas extracted and referenced. Conclusion The higher incidence of infections when using kinase inhibitors is an important consideration, however the number and range inhibitors and their clinical indications are likely to expand, but their exact role in transplantation is yet to be determined. ==========================================================
ACCEPTED MANUSCRIPT Introduction Clinical outcome following solid organ transplantation has significantly improved over the last two decades. However immunosuppressive regimes have unfortunately not evolved at the same pace, with the use of agents first described in the 1960s still being used today.
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Current immunosuppression protocols typically consist of a combination of a calcineurin inhibitor (cyclosporine or tacrolimus - the current cornerstone of immunosuppression), an inosine-5’-mono phosphate dehydrogenase inhibitor, and induction agents such as Alemtuzumab, Basiliximab, or Thymoglobulin.1,2,3
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When these agents were developed as immunosuppressants and adopted into clinical practice, our understanding of allorecognition and alloreactivity was comparatively limited. Consequently, immunosuppression dependent complications now represent one of the primary obstacles to successful transplantation, and this has provided the stimulus for the development of new immunosuppressive drugs of at least equivalent efficacy but improved safety profiles.
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In this review, we consider the role of the kinase family as a promising therapeutic target in transplantation as both as an anti-inflammatory and immunosuppressive agent, summarising relevant studies on kinase inhibitors that have been reported to date for use in solid organ transplantation. Kinase Overview
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There are 518 kinases in the human genome, divided into eight groups. Tyrosine kinases are one major subgroup of this large class of protein kinases; another important subgroup attaches the phosphate moiety to other amino acids (serine and threonine). Amongst the protein kinases, tyrosine kinases have attracted the greatest clinical attention so far, especially in oncology and rheumatology 8, 9
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The tyrosine kinase subfamily consists of two major groups. The receptor kinases (RK) contain an extracellular ligand-binding domain that is connected to the cytoplasmic catalytic domain by a transmembrane helix, and are activated by various growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).8,9,10
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Conversely, non-receptor cytoplasmic kinases (NRK) lack an extracellular component and are located in the cytosol, the nucleus and the inner surface of the plasma membrane. Major NRKs are those linked to sarcoma (SRC), Janus-associated kinase (JAK) and tyrosine kinase from oncogenic transcript that results from fusion of the ABL gene and breakpoint cluster region gene (BCR-ABL).10 The complex pathways exploited by these kinase inhibitors can be summarised here. (see figure one). Janus Kinase as a potential therapeutic target In the context of the immune response, the NRTK which has been studied most intensively to date is JAK, so named after the two-faced Roman god Janus, because JAK uniquely contains two almost identical phosphate-transferring domains but with two distinct and different functions; one domain exhibits the kinase activity while the other negatively regulates the kinase activity of the first.11 The JAK family in mammals consists of four members, namely JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2). The activation of JAK occurs by a ligand–receptor interaction that results in signalling through the phosphorylation of cytokine receptors and the creation of docking sites for a family of signalling proteins known as signal transducers and activators of transcription (STATs).
ACCEPTED MANUSCRIPT Phosphorylated STATs dimerise and then migrate to the nucleus where they become part of transcriptional regulatory machinery and lead to transcription of responsive genes. Under normal physiological conditions the two phosphate-transferring domains ensure that liganddependent activation of the JAK/STAT signalling is transient and tightly regulated. 12
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Signalling via cytokine and growth factor binding to their corresponding receptors on the target cell results in activation of JAKs, which phosphorylate and activate STAT pathways. In addition, STAT can be activated by SRC, ABL and EGFR. The importance of JAKs in vivo was first identified in knockout mice models and through demonstration of JAK3 mutations in patients with immunodeficiency. Mutation of JAK3 results in a severe combined immunodeficiency (SCID) characterised by an almost complete absence of T cells, NK cells and defective B cells.13
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In contrast with other ubiquitously expressed JAKs, JAK3 is primarily expressed in haematopoietically derived cells where it is associated with the IL-2 receptor common γ chain and mediates signalling by IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21; these being the cytokines that are critical for the development and maturation of T cells. The clinical phenotype selectively associated with JAK3 deficiency has led to the suggestion that targeting JAKs might be a strategy for the development of a new class of immunomodulatory drugs.13, 14
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Tofacitinib Formerly designated CP-690,550, tofacitinib was one of the first JAK inhibitors to enter the transplant arena. It was designed to inhibit JAK1 and JAK3 and, to a lesser extent, JAK2, having little effect on TYK2. As a result tofacitinib potently inhibits downstream signalling following common gamma chain cytokine receptor ligation, but also blocks signalling via IFN-γ, IL-6 and to a lesser extent, IL-12 and IL-23. Therefore tofacitinib can inhibit both innate and adaptive immune responses required for T cell differentiation and activation.15 The FDA has approved tofacitinib for use in Rheumatoid Arthritis in 2012 under the trade name of Xeljanz. However its use in transplantation has never been pursued despite data suggesting that tofacitinib may have therapeutic value.
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Preclinical Animal Studies: Using a murine heterotopic heart transplant model, treatment with tofacitinib resulted in a dosedependent increase in graft survival. Treatment was delivered for 28 days, and in the high dose groups the median survival times were greater than 60 days compared to 12 days survival for the placebo group. This study suggested that tofacitinib may represent an efficacious immunosuppressant following heart transplantation, albeit in mice . 17 The same group then performed a series of kidney transplants using mixed leukocyte reaction-mismatched, ABO blood group matched cynomolgus monkeys. Animals received either tofacitinib (n = 18) or placebo (n = 3 -one animal died and so was excluded) and were killed at day 90 or earlier in cases of allograft rejection. The mean survival time in animals treated with tofacitinib was significantly longer than that in control animals (53 ± 7 days vs. 7 ± 1 days, P <0.0003) and was positively correlated with drug exposure. However it is important to note that the control group had longer cold ischaemic times and higher rates of MHC mismatch. Four treated animals killed at 90 days were found to have normal renal function and low-grade rejection on final pathology. The occurrence of rejection was significantly delayed in treated animals but was not prevented (46 ± 7 days from transplantation vs. 7 ± 1 days in controls, P <0.0003). This study confirmed the immunosuppressive potential of tofacitinib but showed independently it was unable to prevent rejection and had significant dose dependent side effects.18 Building on this, further pharmacodynamic assessments identified in vitro exposure to tofacitinib resulted in a significant reduction of IL-2- enhanced IFNγ production by T cells, reduced T-cell surface expression of CD25 and CD71, and reduced T-cell proliferative capacities. These results mirrored similar data in animals dosed with tofacitinib, which displayed a significant reduction of natural killer cell and T-cell numbers whereas CD8+ effector memory T-cell populations remained unaffected.19
ACCEPTED MANUSCRIPT The combination of tofacitinib with mycophenolate mofetil (MMF) significantly improved allograft survival, with the mean survival time in animals treated with MMF alone being significantly extended in animals that concurrently received tofacitinib (23 ± 1 days vs. 59.5 ± 9.8 days, P <0.02). 20
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The side effects observed during these studies during high dose administration of tofacitinib included anaemia and polyoma virus nephritis, yet these were not seen when combined with MMF, suggesting a safe and effective combination may have been identified. As tofacitinib has pan JAK activity; inhibition of JAK2 that mediates erythropoiesis is thought to be a contributing factor to the anaemia seen with its use. 18,19,20
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Phase 1 trial The results of the animal studies enabled the initiation of a dose-escalation study in human renal allograft recipients. The safety effects and tolerability on lymphocyte subsets was a key focus as well as assessing the pharmacokinetics of tofacitinib when co-administered with MMF. 28 patients were recruited in 2008; where six received tofacitinib 5 mg twice daily, six patients received 15 mg, 10 patients received 30 mg, and six patients received placebo. 21 The most common adverse events observed in the study included infection and gastrointestinal symptoms (abdominal pain, diarrhoea, dyspepsia, and vomiting). Higher doses of tofacitinib were associated with anaemia and decreased natural killer cell counts (50%), mirroring what had been observed in earlier primate studies, yet numbers of neutrophils, platelets and T helper cells remained unchanged.21
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The effect of tofacitinib following 29 days of 30 mg administration was investigated in eight kidney transplant recipients. In this study key circulating cytokines within the JAK/STAT pathway were reduced compared to the placebo group . IL-2-induced phosphorylated STAT5 expression in YT cells (immortalised human NK ‘like’ cells) was reduced in the presence of serum samples collected from whole blood on day 29 compared with those collected at baseline. Phosphospecific flow cytometry also revealed tofacitinib reduced IL-2-induced phosphorylated STAT5 in CD3+, CD3+CD4+ and CD3+CD8+ subpopulations as well as interferon gamma (IFNγ)
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production by mononuclear cells. However, examination of blood at day 29 again showed persistent anaemia following the administration of tofacitinib, which seemed to be an ongoing problem.22, 23
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Phase 2a trial Despite the increased incidence of anaemia, a randomized pilot study comparing tofacitinib (15 mg and 30 mg, n = 20 in each group) vs tacrolimus (n = 21) in de novo renal allograft recipients was initiated in 2009. Patients additionally received an IL-2 receptor antagonist, MMF and corticosteroids, which are all part of current standard treatment regimes in kidney transplantation.24 Tofacitinib doses were reduced following 6 months of therapy due to high incidence of BK virus nephropathy in patients treated with high dose tofacitinib, and as a result MMF was discontinued in this group. The 6-month biopsy-proven acute rejection rates were one out of 20, four out of 20 and one out of 21 for 15 mg tofacitinib, 30 mg tofacitinib and tacrolimus,groups respectively, demonstrating that low dose tofacitinib had equivalent efficacy to tacrolimus, but was significantly associated with clinically relevant infections at higher doses. 24 BK virus nephropathy developed in four out of 20 patients in the 30 mg twice-daily administed tofacitinib group. The 6-month rates of cytomegalovirus disease were 2 out of 20, 4 out of 20 and 0 out of 21 for the 15 mg, 30mg tofacitinib groups and tacrolimus groups, respectively. 24 The estimated glomerular filtration rate measured was >70 ml/minute at 6 and 12 months for all groups. In the tofacitinib arms, there were modest lipid elevations and a trend toward more frequent anaemia and neutropenia during the first 6 months.25, 26 This data suggested that co-administration of 30 mg BD tofacitinib with MMF was clearly associated with over immunosuppression. At the 15 mg twice-daily dose, the efficacy and safety profile produced equivocal results to the current standard, tacrolimus yet the incidence of infection was higher and more significant as tofacitinib doses were escalated.24
ACCEPTED MANUSCRIPT Phase 2b trial A phase 2b, randomised, multi-centre, partially blinded study was designed to compare a low or high intensity regime of tofacitinib vs cyclosporine in 331 low to moderate risk de-novo kidney transplant patients. All patients also received basiliximab induction, MMF and corticosteroids.
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Two primary non inferiority endpoints were identified as; incidence of biopsy-proven acute rejection at 6 months or biopsy proven rejection meeting serum creatinine criteria at month 6 (serum creatinine increase ≥0.3 mg/dl and ≥20% from pre-rejection baseline) and measured glomerular filtration rate at month 12. Clinical biopsy-proven acute rejection (11%, 7% and 9% respectively) was observed for the high intensity, low intensity and cyclosporine groups, there was no statistical difference between these groups for rejection. Measured glomerular filtration rates were higher at month 12 for both tofacitinib groups versus the cyclosporine group (65 ml/minute, 65 ml/ minute vs. 54 ml/minute) p<0.01. Fewer patients in the tofacitinib arms developed chronic allograft nephropathy at month 12 compared with cyclosporine patients (25%, 24% vs. 48%). Serious infections, particularly cytomegalovirus and BK virus, developed in 45%, 37% and 25% with high dose tofacitinib again showing higher rates of infection, while cyclosporine had the lowest incidence. Additionally, anaemia, neutropenia and post-transplant lympho-proliferative disorder (PTLD) occurred in four patients in the tofacitinib arm. 27
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The authors concluded that tofacitinib remained equivalent to cyclosporine in preventing acute rejection, and is associated with improved renal function and less chronic allograft histological injury, however the high incidence of infection and anaemia which has been a persistent issue, although dose dependent, ultimately led to the termination of in the agents use in transplantation.27, 28
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Spleen Tyrosine Kinase Spleen tyrosine kinase (SYK), along with Zap-70, is a member of the Syk family of tyrosine kinases. These non-receptor cytoplasmic tyrosine kinases share a characteristic dual SH2 domain separated by a linker domain. SYK has a well-characterised role in the intracellular signalling cascade for classic immunoreceptors such as Fc receptors (FcRs) and the B-cell receptor.8 While Syk and Zap-70 are primarily expressed in hematopoietic tissues, there is expression of Syk in a variety of other tissues making them an attractive target within the field of transplantation. Like JAK3, Zap70 deficiency can also cause severe combined immunodeficiency (SCID), but in this case there is preferential loss of CD8+ T cells. For this reason, Zap70 also remains a target but unfortunately, a clinically useful compound has not yet emerged for testing in the transplant setting.29, 30
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Kidney allografts induce strong T-cell and antibody mediated responses which are known to orchestrate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leukocytes excluding mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and several integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but its pharmacological inhibition reducing activation of monocytes/macrophages and neutrophils makes it an attractive target as an immunosuppressant.30, 31 Fostamatinib is an oral pro-drug and potent inhibitor of immunoglobulin E (IgE) and IgG mediated activation of Fc receptor signalling, with its primary target being spleen tyrosine kinase (Syk). It has been used in rheumatoid arthritis and the treatment of a number of leukaemias. 8, 9 Smith et administered Fostamatinib from day 4 to 10 to Wistar Kyoto rats, and reported reduced glomerular crescents by 100% (P < 0.01) compared with controls. When administered from days 7 to 14, the established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function improved when compared to controls (P < 0.001).32 Hence treatment of Fostamatinib reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, as well as reduced renal monocyte chemo-attractant protein-1 (MCP-1) and IL-1β.32, 33 Pamuk et al, demonstrated that fostamatinib is protective against ischaemia reperfusion in a murine model. Mice were fed with food containing fostamatinib (3 g/kg low dose, 5 g/kg high dose or control, n= 6 per group) for 6 days before induction of intestinal ischaemia reperfusion.
ACCEPTED MANUSCRIPT Artificial ischaemia was performed via anaesthesia and clamping of vessels for a period of 30minutes, which in the treatment groups resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local intestinal and remote lung injury and the beneficial effect was associated with reduced IgM and complement 3 depositions in the tissues, and significant reduction of polymorphonuclear cell infiltration. 34
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The Syk inhibitor CC0482417 (Celgene) was reported to reduce renal allograft rejection and histological damage in the rat model. Recipient rats underwent kidney transplantation and were treated with CC0482417, 30 mg/kg twice daily or placebo, from 1 h before surgery until being euthanased 5 days later. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries.35
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Fostamatinib is currently undergoing a phase 1 trial for chronic graft vs. host disease development following allogeneic stem cell transplantation, but results are yet to be published. 36
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Sotrastaurin Sotrastaurin (AEB071) represents a new class of immunosuppressant, an oral inhibitor of protein kinase C (PKC)F, PKCa, and PKCb, which are essential for T and B cell activation and proliferation. In rodents and primates, sotrastaurin prevents allograft rejection and reduces inflammation, but perhaps more importantly has a reduced side effect profile with no evidence of renal failure, diabetes or hypertension.37
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In the first reported clinical trial, de novo renal transplant recipients with immediate graft function were randomized 1:2 to tacrolimus (control,n=44) or sotrastaurin (300 mg b.i.d.; n=81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary endpoint was a composite of treated biopsy-proven acute rejection (BPAR), graft loss, death or lost to follow-up at month 3. The main safety assessment was estimated glomerular filtration rate (eGFR) and modification of diet in renal disease (MDRD) at month 3. Composite efficacy failure at month 3 was higher in the sotrastaurin group compared to control (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p =0.003), which led to early study termination. Median (± standard deviation [SD]) eGFR was higher for sotrastaurin versus control at all time points from day 7 (month 3: 59.0 ± 22.3 vs. 49.5 ± 17.7 mL/min/1.73m2) p = 0.006. The most common adverse events were gastrointestinal disorders (control: 63.6%; sotrastaurin: 88.9%), which led to study-medication discontinuation in two sotrastaurin patients. The study reported a lower degree of efficacy but better renal function with the calcineurin-inhibitor-free regimen of sotrastaurin + MPA versus the tacrolimus-based control.37 Tedesco-Silva et al followed this with a phase two human de novo renal transplant study, which identified recipients with immediate graft function who were randomized 1:2 to tacrolimus (control, n=44) or sotrastaurin (300 mg b.i.d.; n=81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary composite endpoints were treated biopsyproven acute rejection (BPAR), graft loss, death or lost to follow-up at month three. Composite efficacy failure at month three was found to be higher for the sotrastaurin versus control regimen (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p = 0.003), which led to early study termination.38,39 This was followed by an efficacy and safety study of sotrastaurin with tacrolimus to assess dose ranging, in a non-inferiority study in renal transplant recipients. A total of 298 patients were randomized 1:1:1:1 to receive sotrastaurin 100mg BD, sotrastaurin (200 mg BD) + standardexposure tacrolimus (SET) or reduced-exposure tacrolimus (RET) (SET: n = 76; RET: n = 66), or control (SET + mycophenolic acid [MPA, 720 mg BD]; n = 73). All patients received basiliximab and corticosteroids.40, Composite efficacy failure rates at month 12 were 18.8%, 12.4%, 10.9% and 14.0% for the sotrastaurin 100, 200 and 300 mg, and MPA groups, respectively. The median estimated
ACCEPTED MANUSCRIPT glomerular filtration rates were 55.7, 53.3, 64.9 and 59.2 mL/min, respectively. Patients treated with sotrastaurin developed more frequent tachycardia, and were more frequently discontinued due to adverse side effects.39,40
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Further to this, sotrastaurin with tacrolimus was assessed in a 24-month, multicentre, phase II study in de novo liver transplant recipients. A total of 204 patients were randomized (1:1:1:1) to Sotrastaurin 200 mg B.D. + standard-exposure TAC (n = 50) or reduced-exposure TAC (n = 52), Sotrastaurin 300 mg b.i.d. + reduced-exposure TAC (n = 50), or mycophenolate mofetil (MMF) 1 g b.i.d. + standard-exposure TAC (control, n = 52); all with steroids.41
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Owing to premature study termination, treatment comparisons were only conducted at six months, where composite efficacy failure rates were 25.0%, 16.5%, 20.9% and 15.9% respectively showing sotrastaurin groups having a higher incidence of rejection and adverse events.41
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As a result, development of this compound has been halted due to the continued premature study discontinuation, higher incidences of acute rejection, graft loss, and adverse side effects when compared to standard immunosuppression, deeming it an efficacy failure. 39,40 Despite the promise shown by protein kinase C inhibitors, it is clear that T cell activation is not significantly controlled using this mechanism and so the inhibition of this pathway appears to have little clinical benefit.
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Sunitinib Sunitinib is a RTK for gastrointestinal stromal tumour and metastatic renal cell carcinoma. It inhibits PDGF receptors PDGFR-α and -β and VEGF receptors VEGFR-1, −2, −3 at similar concentrations.8
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Sunitinib has been successfully used in the treatment of imatinib-resistant tumours, which may suggest better efficacy. Is has also been shown to be better than imatinib in preventing PDGFR-β phosphorylation. Rintala et al have previously reported that in an experimental kidney transplantation model, inhibition of PDGF with imatinib prevents chronic rejection. They also found Sunitinib to decrease neointimal formation and smooth muscle cell proliferation in a dose dependent manner in the rat kidney model where grafts preserved better renal function. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expression demonstrating that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation.
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Despite these positive results, it is important to note there is a risk of nephrotic syndrome developing with use of this compound and Sunitinib is yet to be trialled in the context of human transplantation, hence the lack of other studies in this area, suggesting possibility of further work42, 43 Kinase Inhibitors in Ischaemia Reperfusion The various mechanisms participating in the development of ischemia reperfusion injury have been extensively reviewed elsewhere. However the result of a prolonged oxygen deprivation promoting tissue hypoxia causes ATP cell depletion, cell swelling and mitochondrial disruption. The inflammatory cascade is complex, but what is evident is the ubiquitous presence of kinasecontrolled mechanisms. This has identified several kinase family members as potential pharmacological targets for the treatment of ischemia reperfusion injury. Much of the work done in this area is experimental using animal models, which we have summarised below. Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported, where activation of two calcium-insensitive isoforms of the novel PKC subfamily, delta and epsilon, play opposing roles in ischaemia/reperfusion injury. Activation of delta PKC during reperfusion induces cell death through the regulation of mitochondrial function and induction of apoptosis and oncosis. In contrast, activation of epsilon PKC before ischaemia protects mitochondrial function and diminishes apoptosis and oncosis. Both the delta-PKC inhibitor and epsilon-PKC activator conferred cardioprotection against ischemia/reperfusion injury in transgenic mice expressing these peptides in the heart, and co-expression of both peptides
ACCEPTED MANUSCRIPT exerted greater cardioprotective effects than that obtained by the expression of each peptide alone.44
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Using a rat heart transplant model, Tanaka et al identified that combined treatment with protein kinase C activator and delta-protein kinase C inhibitor reduced ischemia-reperfusion injury and decreased the resulting graft coronary artery disease induced by prolonged ischemia. The group analysed the extent of ischemia-reperfusion injury by measuring superoxide generation, myeloperoxidase activity, tumour necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production, and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and caspase 2, 3, 8, and 9 activity.45
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Outside the field of transplantation, work within myocardial ischaemia has identified the inhibition of Rho-kinase activation in leukocytes. Studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury, are cardio protective and reduce cytokine production, but the role of Rho-kinase in leukocytes during I/R injury is not well understood. 46,47 Kitano et al used mice which were subjected to 30-min ischaemia and reperfusion injury. It was found Rhokinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumour necrosis factor (TNF)-α in leukocytes, compared with saline as the vehicle.48
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During liver transplantation, nonalcoholic steatohepatitis (NASH) aggravates the ischemia reperfusion injury by activating various kinases and subsequently releasing cytokines and chemokines. Yang et al, have identified that sorafenib protects NASH rats from IR injury by interfering with the inflammation, necrosis, and apoptotic responses which cause leukocytedependent hepatic microcirculatory dysfunction. The hepatoprotective effects of sorafenib seem to work through the inhibition of the Rho-kinase-dependent Raf/MEK/ERK pathway, which is up regulated during IR injury in the livers of NASH rats.49
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In animal models of lung ischemia–reperfusion and acute inflammatory lung injury, mitogenactivated protein kinases (p38, c-jun N-terminal kinase, extracellular signal–regulated kinase) are activated after oxidative stress. Wolf et al has shown using an experimental rat model that the inhibition of p38 and c-jun kinase provided significant protection from injury.50 Similar animal studies have been performed in liver transplant rat models where Sotraustaurin prevented T lymphocyte activation via a calcineurin-independent pathway, which is of particular appeal as it has also been documented that the role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI) is critical. 51 This study explored the cytoprotective functions of sotraustaurin in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from SD rats were stored for 30h at 4° in UW solution, and then transplanted to syngeneic recipients. Sotraustaurin treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. Sotraustaurin treatment decreased activation of T cells and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished NF-κB/ERK signalling, depressed pro-apoptotic cleaved caspase-3, yet upregulated anti-apoptotic Bcl-2/Bclxl and hepatic cell proliferation. 51 Young Dong et al, who investigated the use of Nafamostat mesilate which inhibits various serine proteases and mitogen activate protein kinase (MAPK) generated during inflammation, has recently made the leap from these experimental animal studies to a single arm pilot study. Nafamostat meslilate is widely used in Asia as an anti-inflammatory and anticoagulant agent. In this study, 10 human livers were flushed with a solution consisting of nafamostat mesilate following graft perfusion using standard HTK solution; these then were then transplanted into 10 consenting patients. Most patients received an emergency deceased donor liver, with mean cold and warm ischemic times of 373 (+/-) and 35 (+/-) minutes, respectively. Due to the limited sample size, a relevant statistical analysis was not possible. However the authors observed decreased mean aspartate aminotransferase (AST) and alanine aminotransferase
ACCEPTED MANUSCRIPT (ALT) levels on the first and third postoperative day as well as lower peak AST and ALT levels, suggesting the impact of ischemia reperfusion had been reduced. 52 Conclusion
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The use of kinase inhibitors has expanded well beyond malignancies to encompass autoimmune disease and many other applications. The role of kinase inhibitors in transplantation remains precarious; but despite their variable safety profile and adverse side effects, protein kinase inhibitors continue to have a role in immune disorders, but the initial use as a potential immunosuppressant in transplantation has shown to be troubled. However, optimising the dose and duration of treatment needed to achieve optimal immunosuppression combined with improved safety may allow this class of medication to be reconsidered in the future.
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Perhaps more importantly, the use of kinase inhibitors may be of benefit in the amelioration of ischaemia reperfusion rather than long-term immunosuppression, capitalising on the beneficial effects of widespread effect without the issues faced of infection associated with longer-term use.
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The number of kinase inhibitors and the range of their clinical indications are likely to expand dramatically in the immediate future. Precisely how these drugs are used in combination with or in place of other therapies such as biologics, steroids and many others remains to be determined. References
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19. Paniagua R, Si MS, Flores MG, Rousvoal G, Zhang S, Aalami O, Campbell A, Changelian PS, Reitz BA, Borie DC: Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allograft. Transplantation 2005, 80:1283-1292. 20. Borie DC, Larson MJ, Flores MG, Campbell A, Rousvoal G, Zhang S, Higgins JP, Ball DJ, Kudlacz EM, Brissette WH, Elliott EA, Reitz BA, Changelian PS: Combined use of the JAJ3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates. Transplantation 2005, 80:1756-1764. 21. Van Gurp E, Weimar W, Gaston R, Brennan D, Mendez R, Pirsch J, Swan S, Pescovitz MD, Ni G, Wang C, Krishnaswami S, Chow V, Chan G: Phase 1 dose escalation study of CP690,550 in stable renal allograft recipients: preliminary findings of safety, tolerability, effects on lymphocyte subsets and pharmacokinetics. Am J Transplant 2008, 8:17111718. 22. Quaedackers ME, Mol W, Korevaar SS, van Gurp EA, vanIjcken WF, Chan G, Weimar W, Baan CC: Monitoring of the immune modulatory effect of CP-690,550 by analysis of the JAK/STAT pathway in kidney transplant patients. Transplantation 2009, 88:1002-1009. 23. Van Gurp E, Schoordijk-Verschoor W, Klepper M, Korevaar SS, Chan G, Weimar W, Baan CC: The eff ect of the JAK inhibitor CP-690,550 on peripheral immune parameters in stable kidney allograft patients. Transplantation 2009, 87:79-86.
ACCEPTED MANUSCRIPT 24. Busque S, Leventhal J, Brennan DC, Steinberg S, Klintmalm G, Shah T, Mulgaonkar S, Bromberg JS,Vincenti F, Hariharan S, Slakey D, Peddi VR, Fisher RA, Lawendy N, Wang C, Chan G: Calcineurin-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients. Am J Transplant 2009, 9:1936-1920.
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25. Wojciechowski D, Vincenti F: Targeting JAK3 in kidney transplantation: current status and future options. Curr Opin Organ Transplant 2011, 16:614-619. 26. Sewgobind VD, Quaedackers ME, van der Laan LJ, Kraaijeveld R, Korevaar SS, Chan G, Weimar W, Baan CC: The jak inhibitor CP-690,550 preserves the function of CD4CD25FoxP3 regulatory T cells and inhibits effector T cells. Am J Transplant 2010, 10:1785-1795.
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27. Vincenti F, Tedesco-Silva H, Busque S, O’Connell P, Friedewald J, Cibrik D, Budde K, Yoshida A, Cohney S, Weimar W, Kim YS, Lawendy N, Lan SP, Kudlacz E, Krishnaswami S, Chan G: Randomized phase 2b trial of tofacitinib (CP-690,550) in de novo kidney transplant patients: effi cacy, renal function and safety at one year. Am J Transplant 2012, 12:2446-2456.
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28. Borie DC, O’Shea JJ, Changelian PC: JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants. Trends Mol Med 2004, 10:532-541. 29. Chan AC, van Oers NS, Tran A. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J. Immunol.1994;152:4758–4766.
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33. Bahjat FR, Pine PR, Reitsma A. An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus. Arthritis Rheum. 2008;58:1433–1444. 34. Pamuk O, Lapchak P, Rani P, Pine P, Lucca J, Tsokos G. Spleen tyrosine kinase inhibition prevents tissue damage after ischaema-reperfusion. Am J Physiol Gastrointest Liver Physiol. 2010; 299(2):G391-G399. 35. Chandran S, Tesch G, Han Y, Woodman N, Mulley W, Kanellis J, Blease K, Ma F, NikolicPaterson D. Spleen tyrosine kinase contributes to acute renal allograft rejection in the rat. Int J Exp Pathol. 2015; 96(1):54-62 36. https://clinicaltrials.gov/ct2/show/NCT02611063 37. Trotter JF, Levy G. Sotrastaurin in Liver Transplantion: Has it had a fair trial. Am J Transpl 2015; 15:1137-1138 38. Friman S, Arns W, Nashan B, Vincenti F, Banas B, Budde K, Cibrik D, Chan L, Klempnauer J, Mulgaonkar S, Nicholson M, Wahlberg J, Wissing K-M, Abrams K, Witte S, Woodle ES. Sotrastaurin, a novel small molecule inhibiting protein-kinase C: randomized phase II study in renal transplant recipients. Am J Transplant. 2011;11:1444–55
ACCEPTED MANUSCRIPT 39. Tedesco-Silva H, Kho MM, Hartmann A, Vitko S, Russ G, Rostaing L, Budde K et al. Sotrastaurin in calcineurin inhibitor-free regimen using everolimus in de novo kidney transplant recipients. Am J Transpl 2013; 7:1757-1768
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40. Russ GR, Tedesco-Silva H, Kuypers DR, Cohney S, Langer RM, Witske O, Eris J et al. Efficacy of sotrastaurin plus tacrolimus after de novo kidney transplantation: randomized, phase II trial results. Am J Transpl 2013; 13(7):1746-56 41. Pascher AA, De Simone PB, Pratschke J. Protein kinase C inhibitor sotrastaurin in de novo liver transplant recipients: A randomized phase II trial. Am J Transpl 2015;15(5):1283-92
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42. Rintala JM, Savikko J, Palin N, Rintala SE, Koskinen PK, Von Willebrand E. Oral platelet derived growth factor and vascular endothelial growth factor inhibitor sunitinib prevents chronic allograft injury in experimental transplantation model. Transplantation 2016; 100(1):103-10
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45. Tanaka M, Gunawan F, Terry R, Inagaki K et al. Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators. J Thorac Cardiovasc Surg. 2005; 129(5):1160-7 46. Hamid SA, Bower HS, Baxter GF. Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am J Physiol Heart Circ Physiol. 2007; 292: H2598–2606.
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47. Bao W, Hu E, Tao L, Boyce R, Mirabile R et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res. 2004; 61(3):548-58
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48. Kitano K, Usui S, Ootsuji H, Takashima S-i, Kobayashi D, Murai H, et al. Rho-Kinase Activation in Leukocytes Plays a Pivotal Role in Myocardial Ischemia/Reperfusion Injury. PLoS ONE. 2014; 9(3): e92242. 49. Yang Y, Huang YT, Lee TY, Chan CC et al. Rho-kinase-dependent pathway mediates the hepatoprotective effects of sorafenib against ischaemia/reperfusion liver injury in rats with non-alcoholic steatohepatitis 50. Wolf PS, Merry HE, Farivar AS, McCourtie AS, Mulligan MS. Stress-activated protein inhibition to ameliorate lung ischemia reperfusion injury. J Thorac Cardiovasc Surg. 2008 Apr; 135(4):856 51. Kamo N, Shen X, Ke B, Busuttil R, Kupiec-Weglinski J. Sotrastaurin a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthoptic liver transplantation. Am K Transplant 2011; 11(11): 2499-2507 52. Young-Dong Y, Dong-Sik K, Sung-Won J, Jae-Hyun H. The effect of nadamostat mesilate to minimise ischemic reperfusion injury of the liver following transplantation: a single arm pilot study. TTS abstract 2016. http://www.tts.org/component/tts/?view=presentation&id=182025
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